Circulating miR-200c and miR-141 and outcomes in patients with breast cancer

Research article[Abstract] Background. The deregulation of microRNAs in both tumours and blood has led to the search for microRNAs to indicate the presence of cancer and predict prognosis. We hypothesize the deregulation of miR-200c/miR-141 in the whole blood can identify breast cancer (BC), and could be developed into a prognostic signature. Methods. The expression of miR-200c and miR-141 were examined in bloods (57 stage I-IV BC patients and 20 age-matched controls) by quantitative reverse-transcription PCR. The associations of circulating microRNAs with clinic and pathological characteristics were analysed. Their effects on survival were analysed by the Kaplan-Meier method and Cox regressions. Results. MiR-200c was down regulated (P < 0.0001) in the blood of BC patients, yielded an area under the ROC curve of 0.79 (90% sensitivity, 70.2% specificity) in discriminating BC from controls. Circulating miR-141 was not discriminating. MiR-200c and miR-141 in the blood of BC patients were inversely correlated (P = 0.019). The miR-200c levels were numerically higher in stage IV and tumours with lower MIB-1. MiR-141 was significantly higher in the blood of patients with stage I-III, lymph node metastasis, and HER2 negative tumours. High blood expression of miR-200c and/or low expression of miR-141 was associated with unfavourable overall survival (hazard ratio, 3.89; [95% CI: 1.28-11.85]) and progression-free survival (3.79 [1.41–10.16]) independent of age, stage and hormonal receptors. Conclusions. Circulating miR-200c and miR-141 were deregulated in BC comparing with controls. Furthermore, miR-200c and miR-141 were independent prognostic factors and associated with distinct outcomes of BC patients.Instituto de Salud Carlos III (España); PI06-154

Prognostic impact of disseminated tumor cells and microRNA-17-92 cluster deregulation in gastrointestinal cancer

[Abstract] The presence of tumor cells in the bone marrow (BM) could be relevant to identifying high risk of disease progression and death in gastrointestinal cancer. However, the molecular profile associated with disseminated tumor cells (DTCs) homing to the BM has yet to be defined. MicroRNAs (miRNA) play key roles in cellular processes implicated in cancer. Thus, we investigated in 38 patients with colorectal, gastric or pancreatic cancer whether the presence of BM-DTCs is associated with a specific miRNA tumor profile and analyzed their potential prognostic impact. DTCs were detected by immunocytochemistry and anti-cytokeratin antibodies in 42.1% of the patients. miRNAs were isolated from formalin-fixed, paraffin-embedded tumors. qRT-PCR was used for miRNA profiling. No significant associations were found among DTC detection and miRNA deregulation. Kaplan-Meier curves demonstrated significantly reduced progression-free survival (PFS) and overall survival (OS) in the DTC-positive patients. Although miR-21 was upregulated in 90.6% of the tumors, no associations with outcomes were found. miR-17 and miR-20a (miRNA-17-92 cluster) were upregulated in 33.3 and 42.4%, respectively. Upregulation of both was correlated and found in 30.3%. Univariate analysis shows that increasing values for miR-20a were significantly associated with reduced PFS (HR 1.022; p=0.016) and OS (HR 1.027; p=0.003). In multivariate Cox models, DTC positivity (HR 4.07; p=0.005) and miR-17 overexpression (HR 2.11; p=0.003) were significantly associated with a higher risk of disease progression. The presence of DTCs in the BM (HR 3.98; p=0.010) and a miR-17 overexpression (HR 2.62; p<0.001) were also associated with a risk of death. Our study suggests that the presence of BM-DTCs and the upregulation of the miR-17-92 cluster in tumors are both significant but independent prognostic markers in gastrointestinal cancer patients.Instituto de Salud Carlos III; PI061541Galicia. Conselleria de Innovación, Industria e Comercio; PGIDT01PXI90001PRXunta de Galicia; PS 08/7

Bioinformatics approach to mRNA markers discovery for detection of circulating tumor cells in patients with gastrointestinal cancer

[Abstract] Background: Detection of tumor cells in the blood, or minimal deposits in distant organs as bone marrow, could be important to identify cancer patients at high risk of relapse or disease progression. Quantitative polymerase chain reaction (PCR) amplification of tissue or tumor selective mRNA is the most powerful tool for the detection of this circulating or occult metastatic cells. Our study aims to identify novel gastrointestinal cancer-specific markers for circulating tumor cell detection. Method: Phase I preclinical study was performed by means of computational tools for expression analysis. In silico data were used to identify and prioritize molecular markers highly expressed in gastrointestinal cancers but absent in hematopoietic-derived libraries. Selected genes were evaluated by means of qRT-PCR in gastrointestinal cancer and hematopoietic cell-lines, normal human bone marrows and bloods, tumor tissue, and blood from cancer patients. Results: Novel and known mRNA markers for circulating tumor cell detection in gastrointestinal cancer have been identified. Among all the genes assessed, PKP3, AGR2, S100A16, S100A6, LGALS4, and CLDN3 were selected and assays based on blood qRT-PCR were developed. Reliably qRT-PCR assays for the novel targets plakophilin 3 (PKP3) and anterior gradient-2 (AGR2) to identify blood-borne cells in cancer patients were developed. Conclusions: Novel and known gastrointestinal-specific mRNA markers for circulating tumor cells have been identified through in silico analysis and validated in clinical material. qRT-PCR assay targeted to PKP3 and AGR2 mRNAs might be helpful to detect circulating tumor cells in patients with gastrointestinal cancer

Circulating miR-200c as a diagnostic and prognostic biomarker for gastric cancer

[Abstract] Background. MicroRNAs are aberrantly expressed and correlate with tumourigenesis and the progression of solid tumours. The miR-200 family determines the epithelial phenotype of cancer cells and regulates invasiveness and migration. Thus, we hypothesised that the quantitative detection of the miR-200 family as epithelial-specific microRNAs in the blood could be a useful clinical biomarker for gastric cancer (GC). Methods. We initially validated the expression levels of miR-200a, 200b, 200c and 141 in GC cell lines (n = 2) and blood from healthy controls (n = 19) using real-time quantitative reverse transcription PCR (qRT-PCR). The microarray expression profiles of the miR-200 family in 160 paired samples of non-tumour gastric mucosae and GC were downloaded through ArrayExpress and analysed. MiR-200c was selected for clinical validation. The qRT-PCR prospective assessment of miR-200c was performed using 67 blood samples (52 stage I-IV GC patients and 15 controls); the area under the receiver operating characteristic curve (AUC-ROC) was estimated. The Kaplan-Meier and Breslow-Wilcoxon tests were used to assess the correlation of miR-200c with overall and progression-free survival (OS and PFS). Multivariate analyses were performed using the Cox model. Results. The miR-200c blood expression levels in GC patients were significantly higher than in normal controls (p = 0.018). The AUC-ROC was 0.715 (p = 0.012). The sensitivity, specificity and accuracy rates of 65.4%, 100% and 73.1%, respectively, were observed. The levels of miR-200c in the blood above the cutoff defined by the ROC curve was found in 17.6% of stage I-II GC patients, 20.6% of stage III patients and 67.7% of stage IV patients (p < 0.001). The miR-200c expression levels were not associated with clinical or pathological characteristics or recent surgical procedures. There was a correlation (p = 0.016) with the number of lymph node metastases and the increased expression levels of miR-200c in blood were significantly associated with a poor OS (median OS, 9 vs 24 months; p = 0.016) and PFS (median PFS, 4 vs 11 months; p = 0.044). Multivariate analyses confirmed that the upregulation of miR-200c in the blood was associated with OS (HR = 2.24; p = 0.028) and PFS (HR = 2.27; p = 0.028), independent of clinical covariates. Conclusions. These data suggest that increased miR-200c levels are detected in the blood of gastric cancer patients. MiR-200c has the potential to be a predictor of progression and survival.Instituto de Salud Carlos III; PI061541Xunta de Galicia; PS08/7

Evaluation of Plakophilin-3 mRNA as a biomarker for detection of circulating tumor cells in gastrointestinal cancer patients

[Abstract] Background: This study aims to assess Plakophilin-3 (PKP3) as a surrogate biomarker of circulating tumor cells in patients with gastrointestinal cancer. Methods: The primary aim is to estimate the diagnostic accuracy of PKP3 real-time reverse transcriptase-PCR in blood. Receiver operating characteristic curves were constructed. Correlations between the blood PKP3 levels and the clinicopathologic features of the study subjects were analyzed. Logistic regression was used to predict outcomes based on PKP3. Results: Sixty-four patients with gastrointestinal cancer and 23 controls were included. The mean relative PKP3 mRNA expression was 48.45 in cancer patients and 2.8 in controls (P < 0.0001). Comparing the PKP3 levels in patients and controls, the area under the curve was 0.852 (95% confidence interval, 0.76-0.94; P < 0.0001) in receiver operating characteristic analysis. A higher blood level of PKP3 mRNA was associated with a more advanced stage (P = 0.025), pT3-4 tumors (P = 0.028), metastasis (P = 0.021), and residual (R2) disease (P = 0.037). Higher PKP3 mRNA was associated with the risk of cancer progression and death (odds ratio, 3.875; 95% confidence interval, 1.781-8.430; P = 0.001). Conclusions: Increased PKP3 mRNA was detected in the blood of gastrointestinal cancer patients. Significant correlations were found with advanced stage, pT3-4, metastatic disease, and the residual disease status. PKP3 mRNA in blood was associated with the risk of cancer progression and death.Rede Galega de Investigación sobre Cancro Colorrectal; Grant 509025250

Evaluation of the Adenocarcinoma-Associated Gene AGR2 and the Intestinal Stem Cell Marker LGR5 as Biomarkers in Colorectal Cancer

We aim to estimate the diagnostic performances of anterior gradient homolog-2 (AGR2) and Leucine-rich repeat-containing-G-protein-coupled receptor 5 (LGR5) in peripheral blood (PB) as mRNA biomarkers in colorectal cancer (CRC) and to explore their prognostic significance. Real-time PCR was used to analyze AGR2 and LGR5 in 54 stages I-IV CRC patients and 19 controls. Both mRNAs were significantly increased in PB from CRC patients compared to controls. The area under the receiver-operating characteristic curves were 0.722 (p = 0.006), 0.376 (p = 0.123) and 0.767 (p = 0.001) for AGR2, LGR5 and combined AGR2/LGR5, respectively. The AGR2/LGR5 assay resulted in 67.4% sensitivity and 94.7% specificity. AGR2 correlated with pT3–pT4 and high-grade tumors. LGR5 correlated with metastasis, R2 resections and high-grade. The progression-free survival (PFS) of patients with high AGR2 was reduced (p = 0.037; HR, 2.32), also in the stage I-III subgroup (p = 0.046). LGR5 indicated a poor prognosis regarding both PFS (p = 0.007; HR, 1.013) and overall survival (p = 0.045; HR, 1.01). High AGR2/LGR5 was associated with poor PFS (p = 0.014; HR, 2.8) by multivariate analysis. Our findings indicate that the assessment of AGR2 and LGR5 in PB might reflect the presence of circulating tumor cells (CTC) and stem cell like CTC in CRC. Increased AGR2 and LGR5 are associated with poor outcomes

Basin-wide variations in Amazon forest structure and function are mediated by both soils and climate

Forest structure and dynamics vary across the Amazon Basin in an east-west gradient coincident with variations in soil fertility and geology. This has resulted in the hypothesis that soil fertility may play an important role in explaining Basin-wide variations in forest biomass, growth and stem turnover rates. Soil samples were collected in a total of 59 different forest plots across the Amazon Basin and analysed for exchangeable cations, carbon, nitrogen and pH, with several phosphorus fractions of likely different plant availability also quantified. Physical properties were additionally examined and an index of soil physical quality developed. Bivariate relationships of soil and climatic properties with above-ground wood productivity, stand-level tree turnover rates, above-ground wood biomass and wood density were first examined with multivariate regression models then applied. Both forms of analysis were undertaken with and without considerations regarding the underlying spatial structure of the dataset. Despite the presence of autocorrelated spatial structures complicating many analyses, forest structure and dynamics were found to be strongly and quantitatively related to edaphic as well as climatic conditions. Basin-wide differences in stand-level turnover rates are mostly influenced by soil physical properties with variations in rates of coarse wood production mostly related to soil phosphorus status. Total soil P was a better predictor of wood production rates than any of the fractionated organic- or inorganic-P pools. This suggests that it is not only the immediately available P forms, but probably the entire soil phosphorus pool that is interacting with forest growth on longer timescales. A role for soil potassium in modulating Amazon forest dynamics through its effects on stand-level wood density was also detected. Taking this into account, otherwise enigmatic variations in stand-level biomass across the Basin were then accounted for through the interacting effects of soil physical and chemical properties with climate. A hypothesis of self-maintaining forest dynamic feedback mechanisms initiated by edaphic conditions is proposed. It is further suggested that this is a major factor determining endogenous disturbance levels, species composition, and forest productivity across the Amazon Basin. © 2012 Author(s). CC Attribution 3.0 License

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030