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Tumour mutational status is an important determinant of the response of metastatic colorectal cancer to targeted treatments. However, the genotype of the tissue obtained at the time of diagnosis might not accurately represent tumour genotype after multiple lines of treatment. This retrospective exploratory analysis investigated the clinical activity of regorafenib in biomarker subgroups of the CORRECT study population defined by tumour mutational status or plasma protein levels. We used BEAMing technology to identify KRAS, PIK3CA, and BRAF mutations in DNA obtained from the plasma of 503 patients with metastatic colorectal cancer who enrolled in the CORRECT trial. We quantified total human genomic DNA isolated from plasma samples for 503 patients using a modified version of human long interspersed nuclear element-1 (LINE-1) quantitive real-time PCR. We also measured the concentration of 15 proteins of interest—angiopoietin 2, interleukin 6, interleukin 8, placental growth factor, soluble TIE-1, soluble VEGFR1, VEGF-A, VEGF-C, VEGF-D, VEGF-A isoform 121, bone morphogenetic protein 7, macrophage colony-stimulating factor, stromal cell-derived factor-1, tissue inhibitor of metalloproteinase 2, and von Willebrand factor—in plasma samples from 611 patients. We did correlative analyses of overall survival and progression-free survival in patient subgroups based on mutational status, circulating DNA concentration, and protein concentrations. The CORRECT trial was registered with ClinicalTrials.gov, number NCT01103323. Tumour-associated mutations were readily detected with BEAMing of plasma DNA, with KRAS mutations identified in 349 (69%) of 503 patients, PIK3CA mutations in 84 (17%) of 503 patients, and BRAF mutations in 17 (3%) of 502 patients. We did not do correlative analysis based on BRAF genotype because of the low mutational frequency detected for this gene. Some of the most prevalent individual hot-spot mutations we identified included: KRAS (KRAS G12D, 116 [28%] of 413 mutations; G12V, 72 [17%]; and G13D, 67 [16%]) and PIK3CA (PIK3CA E542K, 27 [30%] of 89 mutations; E545K, 37 [42%]; and H1047R, 12 [14%]). 41 (48%) of 86 patients who had received anti-EGFR therapy and whose archival tumour tissue DNA was KRAS wild-type in BEAMing analysis were identified as having KRAS mutations in BEAMing analysis of fresh plasma DNA. Correlative analyses suggest a clinical benefit favouring regorafenib across patient subgroups defined by KRAS and PIK3CA mutational status (progression-free survival with regorafenib vs placebo: hazard ratio [HR] 0·52, 95% CI 0·35–0·76 for KRAS wild-type; HR 0·51, 95% CI 0·40–0·65 for KRAS mutant [KRAS wild type vs mutant, pinteraction=0·74]; HR 0·50, 95% CI 0·40–0·63 for PIK3CA wild-type; HR 0·54, 95% CI 0·32–0·89 for PIK3CA mutant [PIK3CA wild-type vs mutant, pinteraction=0·85]) or circulating DNA concentration (progression-free survival with regorafenib vs placebo: HR 0·53, 95% CI 0·40–0·71, for low circulating DNA concentrations; HR 0·52, 95% CI 0·40–0·70, for high circulating DNA concentrations; low vs high circulating DNA, pinteraction=0·601). With the exception of von Willebrand factor, assessed with the median cutoff method, plasma protein concentrations were also not associated with regorafenib activity in terms of progression-free survival. In univariable analyses, the only plasma protein that was associated with overall survival was TIE-1, high concentrations of which were associated with longer overall survival compared with low TIE-1 concentrations. This association was not significant in multivariable analyses. BEAMing of circulating DNA could be a viable approach for non-invasive analysis of tumour genotype in real time and for the identification of potentially clinically relevant mutations that are not detected in archival tissue. Additionally, the results show that regorafenib seems to be consistently associated with a clinical benefit in a range of patient subgroups based on mutational status and protein biomarker concentrations. Bayer HealthCare Pharmaceuticals.

BackgroundThe absence of high-quality next-generation sequencing (NGS) reference material (RM) has impeded the clinical use of liquid biopsies with plasma cell-free DNA (cfDNA) in China.ObjectiveThis study aimed to develop a national RM panel for external quality assessment and performance evaluation during kit registration of non-small-cell lung cancer (NSCLC)-related Kirsten rat sarcoma viral oncogene (KRAS)/neuroblastoma ras oncogene (NRAS)/epidermal growth factor receptor (EGFR)/B-type Raf kinase (BRAF)/mesenchymal–epithelial transition factor (MET) genetic assays using plasma circulating tumor DNA (ctDNA).MethodsMutation cell lines detected by NGS and validated by Sanger sequencing were selected to establish the RM. Cell line genomic DNA was sheared and used to spike basal plasma cfDNA at 10% concentration. Then, the calibration accuracy was determined by four sequencing platforms. Average values were adopted and diluted to 0.1%, 0.3%, 1% and 3% concentrations with basal plasma as the RM panel. Then, five manufacturers were invited to evaluate the performance of the RM panel.Results20 cell lines with 23 clinically important mutations were selected, including six mutations in KRAS, two mutations in NRAS, three in BRAF, four in phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), six in EGFR, one EGFR Gain (4-5 copy) and one MET Gain (2-5 copy). The RM panel consisted of 87 samples, including these 21 mutations at four concentrations (0.1%, 0.3%, 1% and 3%), one MET gain, one EGFR gain and one wild type. The detection rate was 100% for the 3%, 1% and 0.3% samples at all five companies. For the 0.1% concentration, 15 samples had inconsistent results, but at least three companies had correct results for each mutation.ConclusionRM for a KRAS/NRAS/EGFR/BRAF/MET mutation panel for plasma ctDNA was developed, which will be essential for quality control of the performance of independent laboratories.

Acute kidney injury, a common complication of cardiac surgery with cardiopulmonary bypass, is associated with increased morbidity and mortality. Ischemic preconditioning at a remote site mitigates ischemia–reperfusion injury and may prevent acute kidney injury after cardiac surgery, thus providing clinical benefit. To further study this, we enrolled 120 adult patients undergoing elective cardiac surgery for whom cardiopulmonary bypass was anticipated in a randomized, single-blind, and controlled pilot trial. Patients were stratified for the type of surgery and equally assigned to a control group or to receive remote ischemic preconditioning by an automated thigh tourniquet consisting of three 5-min intervals of lower extremity ischemia separated by 5-min intervals of reperfusion. The primary end point was acute kidney injury defined as an elevation of serum creatinine of ≥0.3mg/dl or ≥50% within 48h after surgery. Fifty-nine patients in each group were analyzed on an intention-to-treat basis. Acute kidney injury occurred in 12 remote ischemic preconditioned and 28 control patients, reflecting an absolute risk reduction of 0.27 and a significantly reduced relative risk due to preconditioning of 0.43. Hence, remote ischemic preconditioning prevents acute kidney injury in patients undergoing cardiopulmonary bypass-assisted cardiac surgery.

Insulin resistance is associated with increased risk for and recurrence of breast cancer. Recently, Wnt1-inducible signaling pathway protein-1 (WISP-1) was reported to impair glucose metabolism and insulin sensitivity. In various cancer tissues, Wnt signaling is upregulated and induces further oncogenic and metastatic activity. However, the effects of exercise on serum levels of WISP-1 and its upstream β-catenin have not been studied in cancer patients. We investigated the effects of exercise training on Wnt signaling and insulin sensitivity in breast cancer survivors (BCS). This single-center trial randomized 46 BCS into either 12-week exercise or control groups (1:1), and included an additional 12 age-matched healthy women. Kinanthropometric parameters, serum Wnt signaling markers, and gluco-lipid profiles were evaluated before and after the intervention. Serum β-catenin and WISP-1 concentrations were significantly higher in BCS than in healthy subjects. There was a positive correlation between β-catenin and WISP-1 levels. Exercise training in BCS significantly reduced body fat and waist circumference and enhanced aerobic and muscular fitness. Exercise decreased β-catenin and WISP-1 levels and improved gluco-lipid profiles. There was a notable correlation between changes in HOMA-IR indexes and serum WISP-1, but not with β-catenin during the exercise intervention. In conclusion, a 12-week community-based exercise intervention resulted in significant reductions in serum β-catenin and WISP-1 levels, accompanied by favorable improvements in body composition, physical fitness, and biochemical parameters in BCS. We also highlight that this is the first report concerning effects of exercise on circulating β-catenin and WISP-1 levels and correlations between WISP-1 and insulin sensitivity, which could be important for determining prognoses for BCS.

Patients with cystic fibrosis (CF) suffer from chronic lung infection and inflammation leading to respiratory failure. Vitamin D deficiency is common in patients with CF, and correction of vitamin D deficiency may improve innate immunity and reduce inflammation in patients with CF. We conducted a double-blinded, placebo-controlled, randomized clinical trial of high-dose vitamin D to assess the impact of vitamin D therapy on antimicrobial peptide concentrations and markers of inflammation. We randomized 30 adults with CF hospitalized with a pulmonary exacerbation to 250 000 IU of cholecalciferol or placebo, and evaluated changes in plasma concentrations of inflammatory markers and the antimicrobial peptide LL-37 at baseline and 12 weeks post intervention. In the vitamin D group, there was a 50.4% reduction in tumor necrosis factor-α (TNF-α) at 12 weeks ( P <0.01), and there was a trend for a 64.5% reduction in interleukin-6 (IL-6) ( P =0.09). There were no significant changes in IL-1β, IL-8, IL-10, IL-18BP and NGAL (neutrophil gelatinase-associated lipocalin). We conclude that a large bolus dose of vitamin D is associated with reductions in two inflammatory cytokines, IL-6 and TNF-α. This study supports the concept that vitamin D may help regulate inflammation in CF, and that further research is needed to elucidate the potential mechanisms involved and the impact on clinical outcomes.

Neutrophil gelatinase–associated lipocalin (NGAL) has been described in chronic heart failure (HF) as marker of tubular damage and renal dysfunction; however, less data are available in patients with acute HF. Because of high rate of acute kidney injury (AKI) development, we aimed to investigate the role of NGAL in predicting early AKI development; second, we compared NGAL with respect to cystatin C, B-type natriuretic peptide (BNP), renal function, and blood urea nitrogen (BUN) for outcome prediction. We measured admission serum NGAL, cystatin C, and BNP in 231 patients affected to acute HF; all patients were submitted to daily creatinine, estimated glomerular filtration rate, and measurement to identify inhospital AKI defined by Risk, Injury, Failure, Loss, End-Stage Kidney Disease and Acute Kidney Injury Network criteria. We also measured admission and discharge estimated glomerular filtration rate, creatinine, and BUN to evaluate their prognostic role during a 6-month follow-up period; 78 patients developed AKI during hospitalization. In these subjects, NGAL levels were significantly increased respect to patients without AKI (295 ± 228 vs 129 ± 108 ng/ml, p <0.001). A cutoff of 134 ng/ml has been related to AKI with good sensibility and specificity (85% and 80%, respectively; area under the curve 0.81, p <0.001). BNP was also mildly increased (1,000 ± 906 vs 746 ± 580 pg/ml, p = 0.03) but not cystatin C. Patients with chronic kidney disease demonstrated higher NGAL levels compared with subjects with preserved renal function (258 ± 249 and 120 ± 77 ng/ml, p <0.001). The receiver-operating characteristic curve analysis demonstrated that increased NGAL values were associated with increased mortality (cutoff 170 ng/ml, sensibility 60%, specificity 82%, accuracy 71%, area under the curve 0.77, p <0.001). The same significant correlation was also found for BUN at discharge (cutoff 100 mg/dl, sensibility 65%, specificity 85%, accuracy 71%, area under the curve 0.77, p <0.001). Multivariable Cox regression analysis showed that cutoff 170 ng/ml was related with adverse outcome (hazard ratio 1.77, confidence interval 1.24 to 2.83, p = 0.01). In conclusion, NGAL measurement is a sensible tool to predict AKI during hospitalization. Elevated NGAL levels appear to be related to BUN increase and post-discharge outcome. This suggests a prognostic role of tubular damage beyond renal dysfunction.

Objectives To analyze kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (N-GAL) excretion post-intravenous contrast enhanced-CT (CE-CT) in patients with chronic kidney disease (CKD). Methods Patients were enrolled in a trial on hydration regimes to prevent contrast-induced acute kidney injury (CI-AKI). Blood and urine samples were taken at baseline, 4 – 6, and 48 – 96 h post CE-CT. Urinary KIM-1 and N-GAL values were normalized for urinary creatinine levels, presented as medians with 2.5 – 97.5 percentiles. Results Of the enrolled 511 patients, 10 (2 %) were lost to follow-up. CI-AKI occurred in 3.9 % of patients (20/501). Median KIM-1 values were 1.2 (0.1 – 7.7) at baseline, 1.3 (0.1 – 8.6) at 4 – 6 h, and 1.3 ng/mg (0.1 – 8.1) at 48 – 96 h post CE-CT ( P  = 0.39). Median N-GAL values were 41.0 (4.4 – 3,174.4), 48.9 (5.7 – 3,406.1), and 37.8 μg/mg (3.5 – 3,200.4), respectively ( P  = 0.07). The amount of KIM-1 and N-GAL excretion in follow-up was similar for patients with and without CI-AKI ( P -value KIM-1 0.08, P -value N-GAL 0.73). Neither patient characteristics at baseline including severe CKD, medication use, nor contrast dose were associated with increased excretion of KIM-1 or N-GAL during follow-up. Conclusion KIM-1 and N-GAL excretion were unaffected by CE-CT both in patients with and without CI-AKI, suggesting that CI-AKI was not accompanied by tubular injury. Key Points • KIM-1 and N-GAL excretion were unaffected by intravenous contrast-enhanced CT (CE-CT) . • Patient or procedure characteristics were not associated with increased KIM-1 or N-GAL excretion . • Performance of CE-CT in CKD patients is likely to be safe .

Background: We investigated the clinical implications of KRAS and BRAF mutations detected in both archival tumor tissue and plasma cell-free DNA in metastatic colorectal cancer patients treated with irinotecan monotherapy. Methods: Two hundred and eleven patients receiving second-line irinotecan (350 mg m −2 q3w) were included in two independent cohorts. Plasma was obtained from pretreatment EDTA blood-samples. Mutations were detected in archival tumour and plasma with qPCR methods. Results: Mutation status in tumor did not correlate to efficacy in either cohort, whereas none of the patients with mutations detectable in plasma responded to therapy. Response rate and disease control rate in plasma KRAS wt patients were 19 and 66% compared with 0 and 37%, in patients with pKRAS mutations, ( P =0.04 and 0.01). Tumor KRAS status was not associated with PFS but with OS in the validation cohort. Plasma BRAF and KRAS demonstrated a strong influence on both PFS and OS. The median OS was 13.0 mo in pKRAS wt patients and 7.8 in pKRAS-mutated, (HR=2.26, P <0.0001). PFS was 4.6 and 2.7 mo, respectively (HR=1,69, P =0.01). Multivariate analysis confirmed the independent prognostic value of pKRAS status but not KRAS tumor status. Conclusion: Tumor KRAS has minor clinical impact, whereas plasma KRAS status seems to hold important predictive and prognostic information.

Although resveratrol has widely been studied for its potential health benefits, little is known about its metabolic effects in humans. Our aims were to determine whether the polyphenol resveratrol improves insulin sensitivity in type 2 diabetic patients and to gain some insight into the mechanism of its action. After an initial general examination (including blood chemistry), nineteen patients enrolled in the 4-week-long double-blind study were randomly assigned into two groups: a resveratrol group receiving oral 2 × 5 mg resveratrol and a control group receiving placebo. Before and after the second and fourth weeks of the trial, insulin resistance/sensitivity, creatinine-normalised ortho-tyrosine level in urine samples (as a measure of oxidative stress), incretin levels and phosphorylated protein kinase B (pAkt):protein kinase B (Akt) ratio in platelets were assessed and statistically analysed. After the fourth week, resveratrol significantly decreased insulin resistance (homeostasis model of assessment for insulin resistance) and urinary ortho-tyrosine excretion, while it increased the pAkt:Akt ratio in platelets. On the other hand, it had no effect on parameters that relate to β-cell function (i.e. homeostasis model of assessment of β-cell function). The present study shows for the first time that resveratrol improves insulin sensitivity in humans, which might be due to a resveratrol-induced decrease in oxidative stress that leads to a more efficient insulin signalling via the Akt pathway.

Multiple myeloma (MM) is characterized by the uncontrolled proliferation of plasma cells. Despite recent treatment advances, it is still incurable as disease progression is not fully understood. To investigate MM and its immune environment, we apply single cell RNA and linked-read whole genome sequencing to profile 29 longitudinal samples at different disease stages from 14 patients. Here, we collect 17,267 plasma cells and 57,719 immune cells, discovering patient-specific plasma cell profiles and immune cell expression changes. Patients with the same genetic alterations tend to have both plasma cells and immune cells clustered together. By integrating bulk genomics and single cell mapping, we track plasma cell subpopulations across disease stages and find three patterns: stability (from precancer to diagnosis), and gain or loss (from diagnosis to relapse). In multiple patients, we detect “B cell-featured” plasma cell subpopulations that cluster closely with B cells, implicating their cell of origin. We validate AP-1 complex differential expression (JUN and FOS) in plasma cell subpopulations using CyTOF-based protein assays, and integrated analysis of single-cell RNA and CyTOF data reveals AP-1 downstream targets (IL6 and IL1B) potentially leading to inflammation regulation. Our work represents a longitudinal investigation for tumor and microenvironment during MM progression and paves the way for expanding treatment options. Clonal evolution in multiple myeloma (MM) needs to be understood in both the tumor and its microenvironment. Here the authors perform single-cell multi-omics profiling of samples from MM patients at different stages, finding transitions in the immune cell composition throughout progression.