Circulating Tumor DNA-Based Genomic Profiling Assays in Adult Solid Tumors for Precision Oncology: Recent Advancements and Future Challenges.

Genomic profiling using tumor biopsies remains the standard approach for the selection of approved molecular targeted therapies. However, this is often limited by its invasiveness, feasibility, and poor sample quality. Liquid biopsies provide a less invasive approach while capturing a contemporaneous and comprehensive tumor genomic profile. Recent advancements in the detection of circulating tumor DNA (ctDNA) from plasma samples at satisfactory sensitivity, specificity, and detection concordance to tumor tissues have facilitated the approval of ctDNA-based genomic profiling to be integrated into regular clinical practice. The recent approval of both single-gene and multigene assays to detect genetic biomarkers from plasma cell-free DNA (cfDNA) as companion diagnostic tools for molecular targeted therapies has transformed the therapeutic decision-making procedure for advanced solid tumors. Despite the increasing use of cfDNA-based molecular profiling, there is an ongoing debate about a 'plasma first' or 'tissue first' approach toward genomic testing for advanced solid malignancies. Both approaches present possible advantages and disadvantages, and these factors should be carefully considered to personalize and select the most appropriate genomic assay. This review focuses on the recent advancements of cfDNA-based genomic profiling assays in advanced solid tumors while highlighting the major challenges that should be tackled to formulate evidence-based guidelines in recommending the 'right assay for the right patient at the right time'.

Rapid genomic profiling of circulating tumor DNA in non-small cell lung cancer using Oncomine Precision Assay with Genexus™ integrated sequencer.

Background: Genomic profiling of tumors from cancer patients facilitates molecular-guided therapy. The turnaround time is one of important issues to deliver results timely for clinical decisions. The Ion Torrent™ Genexus™ Integrated Sequencer automates all next generation sequencing (NGS) workflows and delivers results within a day. Methods: In this study, we conducted a feasibility study to evaluate the detection rate of genomic alterations from cell-free total nucleic acid (cfTNA, containing cfDNA and cfRNA) of 119 non-small cell lung cancer using Oncomine Precision Assay on Genexus™ Integrated Sequencer. Oncomine Precision Assay (OPA) covers actionable mutations, copy number variations and fusion genes and that are applicable for the selection of targeted therapy. cfTNA isolated from plasma (derived from 14 ml of blood) were subjected to the Genexus system for library construction, templating, sequencing, and data analyses. Results: The sequencing resulted in median overall depth of 35,773× and median molecular coverage of 2,192× with cfTNA input ranged from 11 to 36 ng. Among the 119 samples evaluated, we detected at least one genomic alteration in plasma cfTNA of 79 cases (66%). When comparing to standard-of-care testing, the sensitivity and specificity of mutation detection in non-small cell lung cancer related genes using liquid biopsy with Genexus-OPA ranged between 49-67% and 93-100%, respectively. 59% of actionable mutations, which were present in tumor tissues, were detected by the Genexus- Oncomine Precision Assay using plasma cfTNA. Among the 5 mutations detected from liquid biopsy only, three mutations are of level 1 evidence according to OncoKB database, highlighting the clinical utilities of liquid biopsy in addressing tumor heterogeneity. Extrathoracic metastasis and levels of lactate dehydrogenase (LDH), C-reactive protein (CRP) and Carcinoembryonic Antigen (CEA) are found to be associated with increased circulating tumor DNA detection. Conclusions: The Genexus™ Integrated Sequencer system is an automated, accurate NGS system with short turnaround time (TAT) that could assist clinicians to make more timely decision.

Lymphocytes in tumor-draining lymph nodes co-cultured with autologous tumor cells for adoptive cell therapy.

BACKGROUND: Tumor-draining lymph nodes (TDLNs) are primary sites, where anti-tumor lymphocytes are primed to tumor-specific antigens and play pivotal roles in immune responses against tumors. Although adoptive cell therapy (ACT) using lymphocytes isolated from TDLNs were reported, characterization of immune activity of lymphocytes in TDLNs to tumor cells was not comprehensively performed. Here, we demonstrate TDLNs to have very high potential as cell sources for immunotherapy. METHODS: Lymphocytes from TDLNs resected during surgical operation were cultured with autologous-tumor cells for 2 weeks and evaluated tumor-reactivity by IFNγ ELISPOT assay. We investigated the commonality of T cell receptor (TCR) clonotypes expanded by the co-culture with tumor cells with those of tumor infiltrating lymphocytes (TILs). RESULTS: We found that that TCR clonotypes of PD-1-expressing CD8+ T cells in lymph nodes commonly shared with those of TILs in primary tumors and lymphocytes having tumor-reactivity and TCR clonotypes shared with TILs could be induced from non-metastatic lymph nodes when they were co-cultured with autologous tumor cells. CONCLUSION: Our results imply that tumor-reactive effector T cells were present even in pathologically non-metastatic lymph nodes and could be expanded in vitro in the presence of autologous tumor cells and possibly be applied for ACT.

Serial circulating tumor DNA monitoring of CDK4/6 inhibitors response in metastatic breast cancer.

Cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) significantly improve progression-free survival and have become the standard therapy for estrogen receptor-positive/human epidermal growth factor receptor 2-negative metastatic breast cancer patients. Treatment surveillance by radiological imaging has some limitations in detection and repeated biopsy genomic profiling is not clinically feasible. Serial circulating tumor DNA (ctDNA) analysis may provide insights into treatment response. Here we performed serial ctDNA analysis (n = 178) on 33 patients. Serial ctDNA analysis identified disease progression with sensitivity of 75% and specificity of 92%. In eight of 12 patients (61%) responding to CDK4/6i who eventually developed progressive disease, serial sampling every 3 or 6 months captured the initial rise of ctDNA with an average lead time of 3 months. In three of eight patients that did not respond to CDK4/6i (progressive disease at first radiological assessment, 3 months), biweekly sequencing within the first cycle of CDK4/6i treatment (1 month) detected sustained ctDNA levels (≥0.2% variant allele frequency), with lead time of 2 months. Serial ctDNA analysis tracked RECIST response, including clinically challenging scenarios (bone metastases or small-sized target lesions), as well as detecting acquired genetic alterations linked to CDK4/6i resistance in the G1 to S transition phase. Circulating tumor DNA analysis was more sensitive than carcinoembryonic antigen or cancer antigen 15-3 serum tumor markers at monitoring tumor response to CDK4/6i treatment. Our findings indicated the possible clinical utility of serial ctDNA analysis for earlier progressive disease detection and real-time monitoring of CDK4/6i response.

Tumor-informed or tumor-agnostic circulating tumor DNA as a biomarker for risk of recurrence in resected colorectal cancer patients.

Introduction: Circulating tumor DNA (ctDNA) has been increasingly recognized as a promising minimally-invasive biomarker that could identify patients with minimal residual disease and a high risk of recurrence after definitive treatment. In this study, we've compared the clinical utility and sensitivity of 2 different approaches to ctDNA analyses: tumor-informed and tumor-agnostic in the management of colorectal (CRC) patients. The clinical benefits of a single timepoint ctDNA analysis compared to serial ctDNA monitoring after definitive treatment were also evaluated to uncover the ideal surveillance protocol. Methods: Patient-paired resected tumor tissues, peripheral blood cells, and a total of 127 pre-operative and serial plasma cell-free DNA (cfDNA) samples after definitive treatment from 38 CRC patients that had undergone curative intent surgery were analyzed using a commercial NGS cfDNA panel. Results: Up to 84% (32/38) of the recruited patients were detected with at least 1 genomic alteration from the tumor tissues that could be monitored using the tumor-informed ctDNA approach and none of the detected alterations were clonal hematopoiesis (CH) related. In contrast, 37% (14/38) of patients were detected with at least 1 monitoring alteration after exclusion of CH mutations using the tumor-agnostic approach. Serial plasma samples after definitive therapy were available for 31 patients. In the landmark ctDNA analysis, 24% (7/29) of patients had detectable ctDNA and were more likely to relapse than ctDNA-negative patients (p < 0.05). The landmark analysis sensitivity and specificity for recurrence were 67% and 87%, respectively. The incorporation of longitudinal ctDNA analysis at 6-months intervals improved the sensitivity to 100%. The median variant allele frequency (VAF) of the ctDNA mutations detected during surveillance was 0.028% (range: 0.018-0.783), where up to 80% (8/10) of the mutations were detected at VAF lower than the tumor-agnostic detection limit of 0.1%. Utilizing the tumor-agnostic approach reduced the recurrence detection sensitivity to 67% (4/6). Serial ctDNA analyses predicted disease recurrence at a median of 5 months ahead of radiological imaging. Conclusion: Longitudinal monitoring using tumor-informed ctDNA testing shows high analytical sensitivity, low probability of false-positive results due to CH mutations, and improved sensitivity in detecting recurrence which may modify the clinical management of CRC.

Application of Nanotechnology in Analysis and Removal of Heavy Metals in Food and Water Resources.

Toxic heavy metal contamination in food and water from environmental pollution is a significant public health issue. Heavy metals do not biodegrade easily yet can be enriched hundreds of times by biological magnification, where toxic substances move up the food chain and eventually enter the human body. Nanotechnology as an emerging field has provided significant improvement in heavy metal analysis and removal from complex matrices. Various techniques have been adapted based on nanomaterials for heavy metal analysis, such as electrochemical, colorimetric, fluorescent, and biosensing technology. Multiple categories of nanomaterials have been utilized for heavy metal removal, such as metal oxide nanoparticles, magnetic nanoparticles, graphene and derivatives, and carbon nanotubes. Nanotechnology-based heavy metal analysis and removal from food and water resources has the advantages of wide linear range, low detection and quantification limits, high sensitivity, and good selectivity. There is a need for easy and safe field application of nanomaterial-based approaches.

Ultradeep targeted sequencing of circulating tumor DNA in plasma of early and advanced breast cancer.

We present a study to evaluate the feasibility and clinical utility of amplicon-based Oncomine Pan-Cancer cell-free assay to detect circulating tumor DNA (ctDNA) in patients with early or advanced breast cancer. In this study, 109 early and metastatic breast cancer patients were recruited before the initiation of treatment. ctDNA mutation profiles were assessed through unique molecular tagging (UMT) and ultradeep next generation sequencing (NGS). For patients with mutations, DNA from corresponding white blood cells (WBC) was sequenced to exclude variants of clonal-hematopoietic (CH) origin. UMT targeted sequencing from plasma of 109 patients achieved a median total coverage of 55 498X and a median molecular coverage of 4187X. Among 53 ctDNA positive samples, 38% were mutation positive by WBC sequencing, indicating potentially false-positive results contributed by CH origin. Prevalence of CH-related mutations was associated with age (P = 7.51 × 10-4 ). After exclusion of CH mutations, ctDNA detection rates were 37% for local or locally advanced breast cancer (stage I-III) and 81% for metastatic or recurrent breast cancer. The ctDNA detection rate correlated with disease stage (P = 2.60 × 10-4 ), nodal spread (P = 6.49 × 10-3 ) and the status of distant metastases (P = 5.00 × 10-4 ). ctDNA variants were detected mostly in TP53, PIK3CA and AKT1 genes, with variants showing therapeutic relevance. This pilot study endorses the use of targeted NGS for non-invasive molecular profiling of breast cancer. Paired sequencing of plasma ctDNA and WBC should be implemented to improve accurate interpretation of liquid biopsy.

Clonal Hematopoiesis in Liquid Biopsy: From Biological Noise to Valuable Clinical Implications.

The use of blood liquid biopsy is being gradually incorporated into the clinical setting of cancer management. The minimally invasive nature of the usage of cell-free DNA (cfDNA) and its ability to capture the molecular alterations of tumors are great advantages for their clinical applications. However, somatic mosaicism in plasma remains an immense challenge for accurate interpretation of liquid biopsy results. Clonal hematopoiesis (CH) is part of the normal process of aging with the accumulation of somatic mutations and clonal expansion of hematopoietic stem cells. The detection of these non-tumor derived CH-mutations has been repeatedly reported as a source of biological background noise of blood liquid biopsy. Incorrect classification of CH mutations as tumor-derived mutations could lead to inappropriate therapeutic management. CH has also been associated with an increased risk of developing cardiovascular disease and hematological malignancies. Cancer patients, who are CH carriers, are more prone to develop therapy-related myeloid neoplasms after chemotherapy than non-carriers. The detection of CH mutations from plasma cfDNA analysis should be cautiously evaluated for their potential pathological relevance. Although CH mutations are currently considered as "false-positives" in cfDNA analysis, future studies should evaluate their clinical significance in healthy individuals and cancer patients.

Plasma or Serum: Which Is Preferable for Mutation Detection in Liquid Biopsy?

BACKGROUND: Blood-based analysis of circulating tumor DNA (ctDNA) is a promising tool for cancer screening, monitoring relapse/recurrence and evaluating response to treatment. Although plasma is widely used to obtain ctDNA, biorepositories worldwide possess a huge number of serum samples and comparative studies on the use of serum vs plasma as ctDNA sources are essential. METHODS: We analyzed cell-free DNA (cfDNA) from matched EDTA-plasma and serum samples from healthy donors and patients with colorectal or lung cancer, and used targeted next-generation sequencing to evaluate mutation detection efficiency and reproducibility. Matched samples from healthy individuals were spiked with reference oligonucleotides and sequenced using the Ion-S5 Oncomine-Pan-Cancer panel. Detection efficiency in matched samples from patients with cancer was evaluated using 2 distinct gene panels and compared to mutations found in tissue-biopsy samples at diagnosis. RESULTS: Mean total cfDNA was 55% higher in serum samples and the presence of longer DNA fragments was significantly increased in serum compared with plasma samples (P = 0.0001 to 0.015). Spiked mutated nucleotides were detected in both samples, but allele frequencies (AF) were approximately half in serum compared with plasma, suggesting ctDNA from serum was more diluted by DNA of noncancerous origins. Matched samples from patients with cancer revealed that up to 44.8% of mutations with low AF were missed in serum samples and concordance rates with somatic mutations found in tissue biopsy at diagnosis was better in plasma samples. CONCLUSION: The use of serum in retrospective studies should consider the limitations for detecting low AF mutations. Plasma is clearly preferable for prospective clinical applications of liquid biopsy.

Clinical significance of clonal hematopoiesis in the interpretation of blood liquid biopsy.

As the use of next-generation sequencing (NGS) for plasma cell-free DNA (cfDNA) continues to expand in clinical settings, accurate identification of circulating tumor DNA mutations is important to validate its use in the clinical management for cancer patients. Here, we aimed to characterize mutations including clonal hematopoiesis (CH)-related mutations in plasma cfDNA and tumor tissues using the same ultradeep NGS assay and evaluate the clinical significance of CH-related mutations on the interpretation of liquid biopsy results. Ultradeep targeted NGS using Oncomine Pan-Cancer Panel was performed on matched surgically resected tumor tissues, peripheral blood cells (PBCs), and 120 plasma cfDNA samples from 38 colorectal cancer patients. The clinical significance of the CH-related mutations in plasma cfDNA was evaluated by longitudinal monitoring of the postoperative plasma samples. Among the 38 patients, 74 nonsynonymous mutations were identified from tumor tissues and 64 mutations from the preoperative plasma samples. Eleven (17%) of the 64 mutations identified in plasma cfDNA were also detected in PBC DNA and were identified to be CH-related mutations. Overall, 11 of 38 (29%) patients in this cohort harbored at least one CH-related mutation in plasma cfDNA. These CH-related mutations were continuously detected in subsequent postoperative plasma samples from three patients which could be misinterpreted as the presence of residual disease or as lack of treatment response. Our results indicated that it is essential to integrate the mutational information of PBCs to differentiate tumor-derived from CH-related mutations in liquid biopsy analysis. This would prevent the misinterpretation of results to avoid misinformed clinical management for cancer patients.

The Roles of Common Variation and Somatic Mutation in Cancer Pharmacogenomics.

Cancer pharmacogenomics is the science concerned with understanding genetic alterations and its effects on the pharmacokinetics and pharmacodynamics of anti-cancer drugs, with the aim to provide cancer patients with the precise medication that will achieve a good response and cause low/no incidence of adverse events. Advances in biotechnology and bioinformatics have enabled genomic research to evolve from the evaluation of alterations at the single-gene level to studies on the whole-genome scale using large-scale genotyping and next generation sequencing techniques. International collaborative efforts have resulted in the construction of databases to curate the identified genetic alterations that are clinically significant, and these are currently utilized in clinical sequencing and liquid biopsy screening/monitoring. Furthermore, countless clinical studies have accumulated sufficient evidence to match cancer patients to therapies by utilizing the information of clinical-relevant alterations. In this review we summarize the importance of germline alterations that act as predictive biomarkers for drug-induced toxicity and drug response as well as somatic mutations in cancer cells that function as drug targets. The integration of genomics into the medical field has transformed the era of cancer therapy from one-size-fits-all to cancer precision medicine.

Immunopharmacogenomics towards personalized cancer immunotherapy targeting neoantigens.

Utilizing the host immune system to eradicate cancer cells has been the most investigated subject in the cancer research field in recent years. However, most of the studies have focused on highly variable responses from immunotherapies such as immune checkpoint inhibitors, from which the majority of patients experienced no or minimum clinical benefit. Advances in genomic sequencing technologies have improved our understanding of immunopharmacogenomics and allowed us to identify novel cancer-specific immune targets. Highly tumor-specific antigens, neoantigens, are generated by somatic mutations that are not present in normal cells. It is plausible that by targeting antigens with high tumor-specificity, such as neoantigens, the likelihood of toxic effects is very limited. However, understanding the interaction between neoantigens and the host immune system remains a significant challenge. This review focuses on the potential use of neoantigen-targeted immunotherapies in cancer treatment and the recent progress of different strategies in predicting, identifying, and validating neoantigens. Successful identification of highly tumor-specific antigens accelerates the development of personalized immunotherapy with no or minimum adverse effects and with a broader coverage of patients.

Worsened arterial stiffness in high-risk cardiovascular patients with high habitual carbohydrate intake: a cross-sectional vascular function study.

BACKGROUND: Previous studies suggested that high dietary carbohydrate intake is associated with increased cardiovascular risk through raised triglyceride and decreased high-density lipoprotein-cholesterol levels. However, the relation between carbohydrate intake and arterial stiffness has not been established. The purpose of this study was to examine this relation among high-risk cardiovascular patients. METHODS: We studied the relation between dietary macronutrient intake and arterial stiffness in 364 patients with documented cardiovascular diseases or risk equivalent (coronary artery diseases 62%, ischemic stroke 13%, diabetes mellitus 55%) and in 93 age-and-sex matched control subjects. Dietary macronutrient intake was assessed using a validated food-frequency questionnaire (FFQ) for Chinese. Heart-ankle pulse wave velocity (PWV) was measured non-invasively with a Vascular Profiling System (VP2000, Colin Corp. USA). A dietary pattern with ≥60% total energy intake derived from carbohydrates was defined as a high-carbohydrate diet according to the Dietary Reference Intakes (DRI) for Chinese. RESULTS: Subjects who consumed a high-carbohydrate diet had significantly higher mean PWV than those who did not consume a high-carbohydrate diet (P = 0.039). After adjustment for potential confounders, high-carbohydrate diet was associated with significantly increased PWV [B = 73.50 (10.81 to 136.19), P = 0.022]. However, there was no significant association between high-carbohydrate diet and PWV in controls (P = 0.634). CONCLUSIONS: High-carbohydrate diet is associated with increased arterial stiffness in patients with established cardiovascular disease or risk equivalent.

Effect of exercise training on vascular endothelial function in patients with stable coronary artery disease: a randomized controlled trial.

BACKGROUND: We aim to investigate the effect of exercise training on endothelial function and exercise capacity in patients with coronary artery disease. METHODS AND RESULTS: A randomized, controlled trial was conducted to determine the effects of an 8-week exercise training programme (n = 32) vs. controls (n = 32) on brachial flow-mediated dilation (FMD) in patients with stable CAD. After 8 weeks, patients received exercise training had significant improvements in FMD (1.84%, p = 0.002) and exercise capacity (2.04 metabolic equivalents, p < 0.001) compared with controls. The change in FMD correlated inversely with baseline FMD (r = -0.41, p = 0.001) and positively with the increase in exercise capacity (r = 0.35, p = 0.005). After adjusting for confounders, every 1 metabolic equivalent increase in exercise capacity was associated with 0.55% increase in FMD. Furthermore, patients received exercise training had significantly increased high-density lipoprotein cholesterol and decreased diastolic blood pressure and resting heart rate compared with controls. However, exercise training did not alter high-sensitivity C-reactive protein, oxidative stress measured as superoxide dismutase and 8-isoprostane, and CD34/KDR + endothelial progenitor cell count. Subgroup analysis showed that FMD was significantly improved only in CAD patients with baseline low exercise capacity (

Effect of herbal consumption on time in therapeutic range of warfarin therapy in patients with atrial fibrillation.

It has been established that herbal intake affects the anticoagulation effects of warfarin, but the long-term impact on anticoagulation control is unclear. We sought to investigate the effect of concomitant herbal intake on anticoagulation control in patients with nonvalvular atrial fibrillation (AF) treated with warfarin. The effects of common herbs were determined by monitoring the international normalized ratio in 250 patients with AF (69 ± 10 years, 50% male). All the patients had been prescribed warfarin therapy for at least 6 months before enrollment, and their dietary intake, including the type and the frequency of common herbs, was recorded using a standardized questionnaire. Up to 50% of the patients reported consumption of foods with herbal ingredients, including garlic (80.4%), ginger (74.8%), green tea (50.4%), and papaya (55.2%) but rarely herbal drugs such as danshen (1.2%), dong guai (0.8%), fenugreek (1.2%), psyllium seed (0.4%), and ginseng (4%). Infrequent users (1 kind of herb for <4 times per week and nonusers) were more likely to have an international normalized ratio within the optimal therapeutic range (2.0-3.0) than frequent users (>1 kind of herb for ≥4 times per week) (58.1% vs 51.1%, P = 0.046). In conclusion, the patients with AF treated with warfarin had little knowledge about the potential interaction of herbal substances in foods with warfarin. The patients who consumed common herbs at least 4 times per week had suboptimal anticoagulation control with warfarin.

Dietary intake of phytoestrogen is associated with increased circulating endothelial progenitor cells in patients with cardiovascular disease.

Endogenous estrogen is known to positively influence the level and functionality of endothelial progenitor cells (EPC). However, the effect of phytoestrogen on EPC is unknown. Isoflavone is a major component of phytoestrogen. This study aims to investigate if the intake of isoflavone has any impact on the circulating level of EPC. We studied 102 consecutive patients (mean age: 66.5 ± 9.5 years, 78% male, all female post-menopausal) with cardiovascular disease (atherothrombotic stroke 62%, coronary artery disease 38%). Circulating levels of CD133(+) EPC were determined by flow cytometry. Non-invasive pulse wave velocity (PWV) was measured. Long-term intake of isoflavone was determined by a validated food frequency questionnaire. Isoflavone intake was positively associated with circulating CD133(+) EPC (r = 0.31, p = 0.001). Patients with circulating CD133(+) EPC <10th percentile had significantly lower isoflavone intake than patients with CD133(+)EPC ≥10th percentile (4.6 ± 3.7 mg/day versus 19.3 ± 30.2 mg/day, p < 0.001). A significant overall linear trend of circulating EPC across increasing tertiles of isoflavone intake was observed (p = 0.004). Adjusted for potential confounders, increased isoflavone intake from the 1st to the 3rd tertile independently predicted increased circulating CD133(+) EPC level by 221 cells/µl (95%CI: 71.4 to 369.8, relative increase 160%, p = 0.004). Gender was not a significant factor (p > 0.05). Furthermore, circulating CD133(+) EPC <10th percentile was independently predictive of increased PWV by 261.7 cm/s (95% CI: 37.1 to 486.2, p = 0.024). The study demonstrated that circulating EPC increased by more than one fold in patients with cardiovascular disease who had higher intake of isoflavone, suggesting that isoflavone may confer vascular protection through enhanced endothelial repair.

Mitochondrial dysfunction induced by statin contributes to endothelial dysfunction in patients with coronary artery disease.

Despite the use of statin therapy, a significant proportion of patients with coronary artery disease (CAD) still develop cardiovascular events. We hypothesized that development of mitochondrial dysfunction (MD) after statin therapy might be linked to endothelial dysfunction and thus limiting its beneficial effects. We studied the effect of MD on endothelial function in 119 patients with CAD on long-term statins (>1 year). Brachial artery flow-mediated dilation (FMD) was assessed by high-resolution ultrasonography and blood levels of lactate, pyruvate, fasting glucose, and lipids were measured. MD (defined by a lactate/pyruvate ratio >75th percentile of the age- and sex-matched normal controls, i.e., > or = 18) was observed in 43/119(36%) patients. There were no significant differences in age, gender, and clinical characteristics between patients with or without MD (all P > 0.05). Patients with MD received higher dose of statin (23.5 +/- 19.3 vs. 17.1 +/- 10.5 mg simvastatin-equivalent dose, P = 0.05) and had lower FMD (2.69 +/- 2.94 vs. 4.33 +/- 2.80%, P = 0.003) than those without MD. Multivariate analysis showed that statin dosage was independently associated with MD (OR:1.03, P = 0.03), and MD significantly predicted an absolute 1.36% decrease in FMD (P = 0.01). In conclusion, a significant proportion of patients with CAD on statin developed MD, which was associated with high-dose statin and with impaired FMD, suggesting that increased statin dosage may induce MD and contribute to endothelial dysfunction in patients with CAD.

Habitual physical activity is associated with endothelial function and endothelial progenitor cells in patients with stable coronary artery disease.

BACKGROUND: Exercise training reduces mortality in patients with coronary artery disease (CAD); however, the impact of habitual physical activity level (PAL) on vascular endothelial function and circulating endothelial progenitor cells (EPCs) remain unknown. METHODS: We assessed habitual PAL using a validated International Physical Activity Questionnaire in 116 patients (67.8+/-9.5 years; 81% male) with stable CAD and preserved left ventricular ejection fraction > or =45%. The number of circulating CD34/KDR+ and CD133/KDR+ EPCs was determined by flow cytometry, and brachial artery flow-mediated dilation (FMD) was measured. RESULTS: The mean PAL of CAD patients with 1644 MET min/week (where MET is metabolic equivalents). With higher habitual PAL tertiles, there were significant linear trends of increased FMD (P = 0.001) and CD133/KDR+ EPCs (P = 0.03), but not of CD34/KDR+ EPCs. Patients with the highest tertile of PAL were associated with an absolute increase of 1.89% in FMD (relative increase 68%, P = 0.003) and 0.12% in CD133/KDR+ EPCs (relative increase 44%, P = 0.01) compared with those in the lowest tertile of PAL, after adjusting for age, sex, presence of hypertension, diabetes mellitus, hypercholesterolemia, smoking, and the use of medications including statins. However, neither CD34/KDR+ nor CD133/KDR+ EPCs significantly correlated with FMD. CONCLUSION: This study showed that higher habitual PAL in patients with CAD was associated with higher FMD and EPC count. Nonetheless, FMD only significantly correlated with increased PAL, but not EPC, suggesting that increased physical activity improves endothelial function through mechanisms other than increasing EPC count.

Daily intake of thiamine correlates with the circulating level of endothelial progenitor cells and the endothelial function in patients with type II diabetes.

Our objective was to determine the relationships between levels of different dietary nutrients intake with circulating endothelial progenitor cells (EPC) and vascular endothelial function in type II diabetic patients. We studied the daily dietary nutrients intake, the numbers of circulating CD34(+)/KDR(+) EPC and CD133(+)/KDR(+) EPC and brachial artery flow-mediated dilation (FMD) in 88 diabetic patients without prior cardiovascular diseases and 91 sex- and age-matched controls. Compared with controls, diabetic patients had lower CD133(+)/KDR(+) EPC count (48.3 +/- 5.2 vs. 84.6 +/- 7.6/microL, p < 0.001), CD34(+)/KDR(+) EPC count (311 +/- 41 vs. 412 +/- 36/microL, p = 0.045), and FMD (2.54 +/- 0.37% vs. 5.46 +/- 0.47%, p < 0.001). After adjusted for age, sex, smoking history, body weight, hemoglobin A1c level, total calorie intake, other dietary vitamin intake, use of antihypertensives, and lipid lowering agents, a higher intake of thiamine was significantly associated with a higher level of circulating CD34(+)/KDR(+) EPC (beta = 0.49, p = 0.028) and CD133(+)/KDR(+) EPC (beta = 0.45, p = 0.037) in diabetic patients, but not in controls. Furthermore, an increased intake of thiamine from 1st to 4th quartile in diabetic patients independently predicted an absolute increase in FMD by 1.29% (p = 0.026, relative increase = 63.5%). This study demonstrated that daily thiamine intake was positively correlated with the circulating number of EPCs and FMD in patients with type II diabetes, independent of other dietary nutrients intake.

Reduction of C-reactive protein with isoflavone supplement reverses endothelial dysfunction in patients with ischaemic stroke.

AIMS: To investigate the effect of oral isoflavone supplement on vascular endothelial function in patients with established cardiovascular disease. METHODS AND RESULTS: A randomized, double-blinded, placebo-controlled trial was performed to determine the effects of isoflavone supplement (80 mg/day, n = 50) vs. placebo (n = 52) for 12 weeks on brachial flow-mediated dilatation (FMD) in patients with prior ischaemic stroke. Compared with controls, FMD at 12 weeks was significantly greater in isoflavone-treated patients [treatment effect 1.0%, 95% confidence interval (95% CI) 0.1-2.0, P = 0.035]. Adjusted for baseline differences in FMD, isoflavone treatment was independently associated with significantly less impairment of FMD at 12 weeks (odds ratio 0.32, 95% CI 0.13-0.80, P = 0.014). The absolute treatment effect of isoflavone on brachial FMD was inversely related to baseline FMD (r = -0.51, P < 0.001), suggesting that vasoprotective effect of isoflavone was more pronounced in patients with more severe endothelial dysfunction. Moreover, isoflavone treatment for 12 weeks resulted in a significant decrease in serum high-sensitivity (hs)-C-reactive protein level (treatment effect -1.7 mg/L, 95% CI -3.3 to -0.1, P = 0.033). Nevertheless, isoflavone did not have any significant treatment effects on nitroglycerin-mediated dilatation, blood pressure, heart rate, serum levels of fasting glucose and insulin, haemoglobin A1c, and oxidative stress as determined by serum superoxide dismutase, 8-isoprostane, and malondialdehyde (all P > 0.05). CONCLUSION: This study demonstrated that 12 week isoflavone treatment reduced serum hs-C-reactive protein and improved brachial FMD in patients with clinically manifest atherosclerosis, thus reversing their endothelial dysfunction status. These findings may have important implication for the use of isoflavone for secondary prevention in patients with cardiovascular disease, on top of conventional interventions.