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OBJECTIVES: There is a growing interest in the relevance of salivary cortisol and cortisone concentrations in stress-related research. To correctly attribute the magnitude of salivary cortisol and cortisone variation as an effect of a stressful event, a coherent understanding of the day-to-day intra-individual and inter-individual variability across the diurnal cycle of the two steroids is required. However, such information is currently lacking. METHODS: This study aimed to overcome these existing limitations by performing an investigation of the biological variation (BV) of salivary cortisol and cortisone within one day and between five days using an LC-MS/MS method. Saliva samples were collected from 20 healthy volunteers immediately after waking up, at 8:00, 12:00, 15:00, 19:00 and 23:00 on each day over five days. All samples were analyzed in duplicate in one run. Nested ANOVA was used to calculate the sums of squares for analytical and biological components of variation. RESULTS: The within-subject BV of salivary cortisol and cortisone (CVI) ranged from a minimum of 29.3 and 19.0â¯% to a maximum of 56.5 and 49.1â¯%, respectively, while the between-subject biological variation (CVG) ranged from 29.7 and 29.0â¯% to 51.6 and 43.6â¯%. The reference change values (RCVs) ranged from 96 to 245â¯% for cortisol and from 55 to 194â¯% for cortisone. A medium index of individuality was observed for both compounds at all time points. CONCLUSIONS: This study provides updated BV estimates and RCVs for different times of day that can be used to assess the magnitude of change in biomarkers in future stress-related research.
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OBJECTIVES: The current study was designed to evaluate the analytical performance of the new Mindray highly sensitive cardiac troponin I (hs-cTnI) chemiluminescent immunoassay on Mindray CL-1200i, as a thorough validation of novel hs-cTnI methods is required before introduction into clinical practice. METHODS: The evaluation of the analytical performance of this hs-cTnI immunoassay encompassed the calculation of the limit of blank (LOB), limit of detection (LOD), functional sensitivity, imprecision, linearity, 99th percentile upper reference limit (URL) and concordance with another previously validated hs-cTnI chemiluminescent immunoassay. RESULTS: The LOB and LOD were 0.32 and 0.35â¯ng/L, whilst the functional sensitivity (expressed as cTnI value with <10â¯% imprecision), was 0.35â¯ng/L. The linearity was excellent throughout a wide range of clinically measurable values (r=1.00 between 0.8 and 9,726.9â¯ng/mL). The intra-assay, inter-assay and total imprecision were 1.1-1.3â¯%, 5.5-8.1â¯% and 5.6-8.2â¯%, respectively. The 99th percentile URL calculated using residual plasma from 246 ostensibly healthy blood donors was 9.2â¯ng/L (4.3â¯ng/L in women vs. 12.3â¯ng/L in men). The Spearman's correlation between Mindray hs-cTnI and Access hs-TnI was 0.97, with mean bias of 7.2â¯% (95â¯% CI, 2.6-11.9â¯%). CONCLUSIONS: Although we failed to confirm the very optimistic analytical characteristics previously reported for this method, our evaluation of the novel Mindray hs-cTnI immunoassay on CL-1200i demonstrated that the overall performance is comparable to that of other commercially available hs-cTnI techniques, making it a viable alternative to other methods.
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Límite de Detección , Troponina I , Humanos , Troponina I/sangre , Troponina I/análisis , Inmunoensayo/métodos , Inmunoensayo/normas , Femenino , Masculino , Adulto , Persona de Mediana Edad , Mediciones Luminiscentes/métodos , Mediciones Luminiscentes/normas , Anciano , Reproducibilidad de los Resultados , Valores de ReferenciaRESUMEN
To investigate the diagnostic performance of relative telomere length (RTL) in cell-free DNA (cfDNA) for endometrioid endometrial cancer (EC). We measured RTL in cfDNA of 40 EC patients (65 ± 12 years) and 31 healthy controls (HC) (63 ± 13 years), excluding in both groups other oncologic and severe non-oncologic diseases to limit confounders. Circulating cfDNA was extracted from serum using the QIAamp DNA Blood Mini kit (Qiagen, Hilden, Germany). After the quantitative real-time polymerase chain reaction, telomere repeat copy number to single-gene copy number ratio was calculated. RTL in cfDNA was found to be significantly lower in EC patients than in HC (p < 0.0001). The diagnostic performance of cfDNA RTL was estimated with receiver operating characteristics (ROC) curve analysis, which showed a diagnostic accuracy for EC of 0.87 (95% CI: 0.79-0.95, p < 0.0001). The cutoff cfDNA RTL value of 2.505 (T/S copy ratio) reported a sensitivity of 80.0% (95% CI: 64.35-90.95) and a specificity of 80.65% (95% CI: 62.53-92.55). Significant differences of RTL among EC stages or grades (p = 0.85 and p = 0.89, respectively) were not observed. Our results suggest that cfDNA RTL analysis may be a diagnostic tool for EC detection since the early stage, whilst its diagnostic performance seems unsatisfactory for cancer progression, staging, and grading. However, further studies are needed to confirm these preliminary findings. In particular, future investigations should focus on high-risk patients (such as those with atypical endometrial hyperplasia) that may benefit from this tool, because TL shortening is not specific for EC and is influenced by other oncologic and non-oncologic diseases.
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Biomarcadores de Tumor/genética , Ácidos Nucleicos Libres de Células/genética , ADN de Neoplasias/genética , Neoplasias Endometriales/diagnóstico , Homeostasis del Telómero , Telómero/genética , Anciano , Biomarcadores de Tumor/sangre , Estudios de Casos y Controles , Ácidos Nucleicos Libres de Células/sangre , Terapia Combinada , ADN de Neoplasias/sangre , Neoplasias Endometriales/genética , Neoplasias Endometriales/terapia , Femenino , Estudios de Seguimiento , Dosificación de Gen , Humanos , Persona de Mediana Edad , PronósticoRESUMEN
A specific subset of micro RNAs (miRs), including miR-133 and miR-206, is specifically expressed in muscle tissue, so that they are currently defined as muscular miRs (myomiRs). To further elucidate the role of myomiRs in muscle biology, we measured miR-133a and miR-206 in plasma of 28 middle-age recreational athletes. The study population consisted of 28 middle aged, recreation athletes (11 women and 17 men; mean age, 46 years) who completed a 21.1 km, half-marathon. The plasma concentration of miR-133a and miR-206, the serum concentration of creatine kinase (CK) and high-sensitivity (HS) cardiac troponin T (cTnT), as well as capillary lactate, were measured before and immediately after the run. The median serum concentration of total CK (257 versus 175 U/L; p < .001), cTnT (17.8 versus 5.6 ng/L; p < .001), and the plasma values of both miR-133a (4.22 versus 0.64 × 10-4; p < .001) and miR-206 (1.36 versus 0.63 × 10-4; p = .001) were considerably increased immediately after the half-marathon run. In multivariate analysis only post-exercise capillary lactate was found to be independently associated with running time. A significant and independent correlation was observed between plasma variations of the two miRs, but not with other physiological or laboratory parameters. The results of this study suggest that the biological significance of miR-133a and 206 variation after middle-distance running parallels but not overlaps the release of biomarkers of nonspecific tissue damage. Enhanced plasma values of these myomiRs may hence reflect a physiological response to high-intensity and/or prolonged exercise rather than tissue injury.
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MicroARNs/genética , Músculo Esquelético/metabolismo , Resistencia Física/fisiología , Carrera/fisiología , Atletas , Creatina Quinasa/sangre , Creatina Quinasa/genética , Femenino , Regulación de la Expresión Génica , Humanos , Ácido Láctico/sangre , Masculino , MicroARNs/sangre , Persona de Mediana Edad , Análisis Multivariante , Troponina T/sangre , Troponina T/genéticaRESUMEN
BACKGROUND: The non-invasive diagnostic approach for early detection of endometrial cancer (EC) remains limited. To date, human epididymis protein 4 (HE4) has been intensively studied but its diagnostic is controversial in EC. DJ-1 is an oncoprotein secreted by cancer cells, recently identified as a potential diagnostic biomarker for breast cancer, melanoma, and pancreatic cancer. The aim of this study was to compare the diagnostic performances of DJ-1 and HE4 measured in EC patients and healthy controls (HC). METHODS: Forty-five patients (63.9±12.0 years) with EC and 29 (63.2±13.3 years) HC were enrolled. Serum concentrations of DJ-1 and HE4 were measured using ELISA kits developed by R&D (Minneapolis, USA) and Fujirebio Diagnostic (Malvern, PA, USA), respectively. Differences between EC patients and HC were assessed by Mann-Whitney test and associations were tested by Spearman's correlation. The diagnostic performance was assessed using receiver operating characteristics (ROC) curves analysis. RESULTS: Serum DJ-1 concentrations were found to be higher in EC patients than in HC (9533.6 vs 1988.5 pg/mL; P<.0001). The area under the ROC curve (ROC-AUC) was 0.95 (P<.0001). At the cut-off of 3654 pg/mL, the sensitivity and specificity were 0.89 and 0.90, respectively. HE4 serum levels were higher in EC patients than in HC (75.3 vs 56.2 pmol/L; P=.019), with an AUC of 0.66 (P=.020). The AUC obtained by the combination of the two markers resulted 0.96 (P<.0001). CONCLUSION: These results suggest that increased serum DJ-1 levels are associated with EC and that this biomarker may be potentially useful for diagnosing EC.
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Biomarcadores de Tumor/sangre , Neoplasias Endometriales/sangre , Neoplasias Endometriales/epidemiología , Proteína Desglicasa DJ-1/sangre , Proteínas/análisis , Anciano , Área Bajo la Curva , Estudios de Casos y Controles , Femenino , Humanos , Persona de Mediana Edad , Proteína 2 de Dominio del Núcleo de Cuatro Disulfuros WAPRESUMEN
BACKGROUND: Endometrial cancer (EC) is currently considered the fourth most frequent female cancer in Europe. In an attempt to achieve an early diagnosis, many studies have identified some putative biomarkers for gynecologic cancers, including circulating microRNAs (miRs) and aberrant promoter methylation status. Previous studies which have investigated miR-203 expression profiles in EC tissues and normal endometrial tissues concluded that miR-203 is regulated by methylation promoter. The aim of this study was to investigate the expression of miR-203 and promoter methylation levels in serum of EC patients and healthy controls (HC). METHODS: Forty-five EC patients (64 ± 12 years) and 30 HC (63 ± 13 years) were enrolled before undergoing therapeutic procedures. RNA extraction from serum was performed with mirVana PARIS Isolation Kit (Thermo Scientific). miR expression was assessed by quantitative RT-PCR (Applied Biosystems). The expression levels of miR were normalized to miR-16 and calculated using the 2-ΔCt method. A quantitative methylation-specific PCR (MSP) technique was used to analyze miR-203 promoter methylation status. Differences between groups were assessed by Mann-Whitney test (for continuous variables) and chi-squared test (for categorical variables), whereas the correlation was calculated using Spearman's test. The diagnostic performance of miR-203 was defined using receiver operator characteristic (ROC) curves. RESULTS: Serum expression levels of miR-203 were higher in EC patients compared to HC (p = 0.002). Aberrant miR-203 methylation was detected in 11/45 (24.4%) EC patients and in 2/30 (6.6%) HC (p = 0.046). The expression levels of miR-203 were not significantly correlated with promoter methylation status. The area under the curve of miR-203 expression was 0.71 (p = 0.002). CONCLUSIONS: The high circulating miR-203 expression levels in EC patients compared to HC confirm the role of this miR as a potential biomarker for diagnosis of EC. Aberrant miR-203 methylation assessed in the peripheral blood does not apparently reflect cancer biology.
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Neoplasias Endometriales/sangre , MicroARNs/sangre , Anciano , Estudios de Casos y Controles , Metilación de ADN , Femenino , Humanos , MicroARNs/genética , Persona de Mediana Edad , Regiones Promotoras GenéticasRESUMEN
OBJECTIVE: Current evidence suggests that no single serum biomarker displays satisfactory diagnostic performance in patients with endometrial carcinoma (EC), the most frequent gynecological cancer in developed countries. However, aberrant tissue microRNA (miRNA) expression has been recently described in EC. Therefore, this study aimed to investigate the differential expression of 4 serum miRNAs and their association with CA125 (cancer antigen 125) and HE4 (human epididymis protein 4) in EC patients and in a control population. METHODS: Forty-six consecutive women with EC and 28 matched control subjects without a history of cancer or other diseases were enrolled. Total serum RNA was extracted using mirVana PARIS Kit. TaqMan MicroRNA Assay was used for quantitative real-time reverse transcriptase-polymerase chain reaction on ABI 7500 Sequence Detection System to assess differential miRNAs expression. The relative expression levels of 4 miRNAs (miR-222, miR-223, miR-186, and miR-204) were normalized to miR-16 and calculated using the 2-â³Ct approach. RESULTS: Serum levels of miR-186, miR-222, and miR-223 appeared to be significantly higher in patients compared with control subjects (P = 0.004, P = 0.002, and P < 0.0001). Contrarily, serum miR-204 was found to be significantly lower in EC patients (P < 0.0001). The diagnostic performance of miRNAs was found to be significantly better than that of CA125. Among the various biomarker tested, serum miR-204 and HE4 exhibited the best diagnostic performance for discriminating EC patients from control subjects. CONCLUSIONS: These results underpin that the 4 miRNAs that we have investigated are implicated in development and progression of EC, thus opening new avenues in EC diagnostics.
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Neoplasias Endometriales/sangre , Neoplasias Endometriales/genética , MicroARNs/sangre , MicroARNs/genética , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/biosíntesis , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/genética , Antígeno Ca-125/sangre , Estudios de Casos y Controles , Femenino , Humanos , Proteínas de la Membrana/sangre , MicroARNs/biosíntesis , Persona de Mediana Edad , Proteínas/metabolismo , Proteína 2 de Dominio del Núcleo de Cuatro Disulfuros WAPRESUMEN
BACKGROUND: Recent evidence suggests that microRNAs play an important role in cancer diagnostics. We assessed plasma microRNA-21 levels in patients with colorectal cancer (CRC) at different stages and in patients with benign polyps. METHODS: Plasma levels of miR-21 were assessed by quantitative reverse transcription polymerase chain reaction assay in plasma samples of 76 CRC patients and in 20 patients with benign polyps. Differences between groups were evaluated with Mann-Whitney and Kruskal-Wallis tests. RESULTS: No significant differences of miR-21 plasma levels were observed between CRC patients and subjects with benign polyps (p > 0.05). Also, no significant differences were found between CRC patients with advanced (III-IV) or early cancer stages (I-II) (p > 0.05). CONCLUSIONS: These results do not support the hypothesis that circulating miR-21 expression is increased in adenoma-carcinoma-advanced carcinoma sequence. Accordingly, plasma miR-21 assessment does not appear to be a useful biomarker for diagnosing and staging CRC.
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Neoplasias Colorrectales/diagnóstico , MicroARNs/sangre , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias Colorrectales/genética , Femenino , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: Although recent advances in circulating DNA analysis allow the prediction of tumor genomes by noninvasive means, some challenges remain, which limit the widespread introduction of cfDNA in cancer diagnostics. We analyzed the status of the two best characterized colorectal cancer (CRC) genetic and epigenetic alterations in a cohort of CRC patients, and then compared the degree to which the two patterns move from tissue to plasma in order to improve our understanding of biology modulating the concordance between tissues and plasma methylation and mutation profiles. METHODS: Plasma and tumor tissues were collected from 85 patients (69±14 years, 56 males). KRAS and SEPT9 status was assessed by allele refractory mutation system quantitative PCR and quantitative methylation-specific PCR, respectively. Six of the most common point mutations at codon 12 and 13 were investigated for KRAS analysis. RESULTS: KRAS mutations and SEPT9 promoter methylation were present in 34% (29/85) and in 82% (70/85) of primary tumor tissue samples. Both genetic and epigenetic analyses of cfDNA revealed a high overall concordance and specificity compared with tumor-tissue analyses. Patients presenting with both genetic and epigenetic alterations in tissue specimens (31.8%, 27/85) were considered for further analyses. The median methylation rates in tumour tissues and plasma samples were 64.5% (12.2-99.8%) and 14.5% (0-45.5%), respectively. The median KRAS mutation load (for matched mutations) was 33.6% (1.8-86.3%) in tissues and 2.9% (0-17.3) in plasma samples. The plasma/tissue (p/t) ratio of SEPT9 methylation rate was significantly higher than the p/t ratio of KRAS mutation load, especially in early stage cancers (p=0.0108). CONCLUSION: The results of this study show a discrepant rate of epigenetic vs. genetic alterations moving from tissue to plasma. Many factors could affect mutation cfDNA analysis, including both presence of tumor clonal heterogeneity and strict compartmentalization of KRAS mutation profile. The present study highlights the importance of considering the nature of the alteration when analyzing tumor-derived cfDNA.
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Neoplasias Colorrectales/genética , Metilación de ADN/genética , Epigénesis Genética/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Septinas/genética , Adenocarcinoma/genética , Anciano , Análisis Mutacional de ADN , ADN de Neoplasias/genética , Femenino , Humanos , Masculino , Mutación Puntual/genética , Regiones Promotoras GenéticasRESUMEN
Patients with Systemic Mastocytosis (SM) need a highly sensitive diagnostic test for D816V detection of the KIT receptor gene. Along with histology/cytology and flow cytometry evaluation, bone marrow (BM) from 110 consecutive adult patients referred with a suspicion of SM to Multidisciplinary Outpatient Clinic for Mastocytosis in Verona were tested both by Amplification Refractory Mutation System Reverse Transcriptase quantitative real time Polymerase Chain Reaction (ARMS-RT-qPCR) and RT-PCR+Restriction Fragment Length Polymorphism (RFLP) followed by Denaturing-High Performance Liquid Chromatography (D-HPLC) and Sanger sequencing. ARMS-RT-qPCR identified D816V mutation in 77 patients, corresponding to 100% of cases showing CD25(+) mast cells (MCs) whereas RT-PCR+RFLP/D-HPLC+sequencing revealed D816V mutations in 47 patients. According to the 2008 WHO criteria 75 SM, 1 Cutaneous Mastocytosis (CM), 1 monoclonal MC activation syndrome (MMAS), and 1 SM Associated with Haematologic Non-Mast Cell Disorder (SM-AHNMD) were diagnosed. Seventeen out 75 SM patients (23%) would have not satisfied sufficient WHO criteria on the basis of the sole RT-PCR+RFLP: these patients had significantly lower serum tryptase levels and amount of CD25(+) MCs. Therefore, ARMS-RT-qPCR might result particularly useful, in patients that do not fulfil major BM histological criterion, for the recognition of indolent SM with a very low MC burden.
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Biomarcadores de Tumor/genética , Médula Ósea/patología , Mastocitosis Sistémica/diagnóstico , Mastocitosis Sistémica/genética , Mutación Puntual/genética , Proteínas Proto-Oncogénicas c-kit/genética , Adulto , Anciano , Anciano de 80 o más Años , Médula Ósea/metabolismo , Cromatografía Líquida de Alta Presión , Femenino , Estudios de Seguimiento , Humanos , Masculino , Mastocitosis Sistémica/sangre , Persona de Mediana Edad , Polimorfismo de Longitud del Fragmento de Restricción , Pronóstico , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Adulto JovenRESUMEN
BACKGROUND: Myeloproliferative neoplasms (MPNs) are clonal myeloid disorders characterized by the overproduction of mature blood cells. The pathogenetic hallmark of MPNs is the dysregulation of JAK-STAT signaling, usually associated with the JAK2 V617F mutation. Multiple additional genetic and epigenetic alterations that constitutively activate the JAK-STAT signaling pathway have been described, including the modulation of the microRNAs (miRs) expression levels. The aims of our study were to investigate JAK2 V617F mutation allele burden and miR-143 expression levels in MPNs patients and to investigate the correlation between these genetic signatures and hematological parameters. METHODS: In total 78 patients with a clinical diagnosis of polycythemia vera (PV), essential thrombocythemia (ET) and idiopathic myelofibrosis (IM), made according to the WHO 2008 criteria, were included in the study. Twenty healthy subjects were checked as controls. Quantification of JAK2 V617F mutation and miR-143 expression levels were determined by real-time quantitative polymerase chain reaction. RESULTS: The miR-143 expression in MPNs patients was 2.97-fold higher than in controls. JAK2 V617F mutation allele burden and miR-143 expression level resulted higher in PV and IM respect to ET patients. Patients who had V617F allele burden >50% displayed a higher miRNA-143 expression level than patients with allele burden <50%. In MPNs patients, a statistically significant positive correlation was observed between JAK2 V617F mutation allele burden and hemoglobin and hematocrit values and between miR-143 expression levels and platelet count. CONCLUSIONS: Our findings of aberrant miR-143 expression support the concept that factors other than JAK2 V617F mutation may contribute to the pathogenesis and some clinical signs of MPNs.