1,720,979 research outputs found
Evaluation of a coated blade spray-tandem mass spectrometry assay as a new tool for the determination of immunosuppressive drugs in whole blood
No full textImmunosuppressive drugs (ISDs) are primarily administered following solid organ transplant or for treatment of a variety of autoimmune conditions. Their principal function is to suppress the activity of the immune system; however, the levels must be carefully monitored due to adverse effects of over- or underadministration. A technology for rapid quantitative screening, named coated blade spray (CBS), was directly coupled to a triple quadrupole mass spectrometer (MS/MS) to measure the concentration of ISDs (i.e., cyclosporine A, tacrolimus, everolimus, sirolimus) in whole blood samples. We evaluated the stability of replicate measurements over a 10-day period (precision), assessed linearity and limit of quantification, and performed a method comparison against a validated clinical immunoassay (Abbott ARCHITECT). Total interday variation of less than 5% for all target compounds at three different concentrations was achieved. The sensitivity of the method was determined as 0.25, 1, 1, and 2.5 ng/mL for everolimus, sirolimus, tacrolimus, and cyclosporine A, respectively. The concentrations of three immunosuppressive drugs in 284 patient samples (i.e., ~ 95 samples of cyclosporine A, tacrolimus, or sirolimus) obtained using the CBS-MS/MS methodology were compared with concentrations previously quantified on an Abbott ARCHITECT immunoassay system. Our analysis demonstrated significant statistical similarities between both methods. The results demonstrate that CBS-MS/MS is a suitable alternative to conventional methodologies for monitoring of ISDs from whole blood in a clinical setting.Fil: Rickert, Daniel A.. University of Waterloo; CanadáFil: Gómez Ríos, Germán A.. University of Waterloo; CanadáFil: Nazdrajic, Emir. University of Waterloo; CanadáFil: Tascon, Marcos. Universidad Nacional de San Martín. Instituto de Investigación e Ingeniería Ambiental. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación e Ingeniería Ambiental; ArgentinaFil: Kulasingam, Vathany. University of Toronto; Canadá. University Health Network; CanadáFil: Pawliszyn, Janusz B.. University of Waterloo; Canad
Identification and Validation of Candidate Breast Cancer Biomarkers: A Mass Spectrometric Approach
Get PDFOne of the best ways to diagnose breast cancer early or to predict therapeutic response is to use serum biomarkers. Unfortunately, for breast cancer, we do not have effective serological biomarkers. We hypothesized that novel candidate tumor markers for breast cancer may be secreted or shed proteins that can be detected in tissue culture supernatants of human breast cancer cell lines. A two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) strategy was utilized to identify and compare levels of extracellular and membrane-bound proteins in the conditioned media. Proteomic analysis of the media identified in excess of 600, 500 and 700 proteins in MCF-10A, BT474 and MDA-MB-468, respectively. We successfully identified the internal control proteins, kallikreins 5, 6 and 10 (ranging in concentration from 2-50 µg/L), as validated by ELISA and confidently identified HER-2/neu in BT474 cells. Sub-cellular localization was determined based on Genome Ontology (GO) for the 1,139 proteins, of which 34% were classified as extracellular and membrane-bound. Tissue specificity, functional classifications and label-free quantification were performed. The levels of eleven promising molecules were measured in biological samples to determine its discriminatory ability for control versus cases. This screen yielded activated leukocyte cell adhesion molecule (ALCAM) as a promising candidate. The levels of ALCAM, in addition to the classical breast cancer tumor markers carbohydrate antigen 15-3 (CA 15-3) and carcinoembryonic antigen (CEA) were examined in 300 serum samples by quantitative ELISA. All three biomarkers effectively separated cancer from non-cancer groups. ALCAM, with area under the curve (AUC) of 0.78 [95% CI: 0.73, 0.84] outperformed CA15-3 (AUC= 0.70 [95% CI: 0.64, 0.76]) and CEA (AUC= 0.63 [95% CI: 0.56, 0.70]). The incremental values of AUC for ALCAM over that for CA15-3 were statistically significant (Delong test, p <0.05). Serum ALCAM appears to be a new biomarker for breast cancer and may have value for disease diagnosis.Ph
Integrating High-throughput Technologies for the Identification and Validation of Novel Ovarian Cancer Biomarkers
Get PDFOvarian cancer is the most lethal gynaecological malignancy in North America. Although survival rates are high when the disease is diagnosed at an early stage, this decreases significantly in late-stage diagnoses. The majority of cases are of epithelial origin, which can be classified into four major subtypes: serous, mucinous, endometrioid and clear cell carcinoma. Unfortunately, the clinically-approved markers for ovarian cancer - CA125 and HE4 alone - perform poorly for the mucinous, endometrioid and clear cell subtypes and thus, diagnosis of these subtypes remains a significant challenge. To this end, an integrated approach to ovarian cancer biomarker discovery was developed in this study through combining proteomics with other high-throughput platforms. Using proteomic analyses of ascites fluid from women with ovarian cancer and conditioned media of ovarian cancer cell lines, 15 high-priority candidates were identified as putative novel biomarkers using said integrated approach. Serum validation revealed two markers - folate receptor 1 and kallikrein 6 - to have comparable diagnostic ability to the clinically-approved markers, albeit with similar limitations in their ability to detect patients of the non-serous histotypes as well. Fortunately, the validation of the two in-house markers served as proof-of-principle of our integrated approach to biomarker discovery. As a result, the approach was employed on non-serous ovarian cancer tissues to identify novel markers specific to the mucinous, endometrioid and clear cell subtypes of ovarian cancer. Over 9000 unique proteins were identified in this exercise and with the use of an unbiased filtering algorithm based on transcriptomics and bioinformatics, a list of high-priority candidates for each subtype was generated. Several of the high-priority candidates have shown strong biological and molecular relevance to their respective subtypes, demonstrating the robustness and utility of the integrated approach. Future studies will need to investigate these candidates in independent serum cohorts to truly assess for their ability to diagnose their respective subtypes.Ph.D.2017-11-30 00:00:0
Individualizing Ovarian Cancer Surveillance: Discovery and Validation of Serological Personalized Biomarkers of Recurrence Using Multiplex Proteomics Technologies
Get PDFIn Canada, ovarian cancer is the third most common female reproductive cancer and the leading cause of deaths among gynecological cancers. Although remission is observed in most ovarian cancer patients after first-line treatment, >80% of advanced cases see recurrence with a median survival of 12-24 months from the time of recurrence. The classical ovarian cancer biomarker, CA125, is controversial for monitoring recurrence as initiating second-line therapy sooner based on CA125 does not impact survival. Furthermore, CA125 is non-elevated at diagnosis in 10-20% of advanced ovarian cancer cases in general, leaving this population with no widely used biomarkers for surveillance. Patients being monitored with CA125 also have a 10-40% chance of CA125 being non-elevated at recurrence. With increasing selection of immunotherapies and precision medicines, novel personalized ovarian cancer biomarkers could help individualize the surveillance process. Due to tumour heterogeneity, we hypothesize that quantifying the unique array of tumour-derived serological proteins with advanced proteomics methods could identify personalized marker signatures that sensitively detect relapse. We first assessed the technical potential of two multiplex proteomics technologies for detecting proteins that may correlate to tumour burden in cancer patients. For our subsequent discovery study, we employed the proximity extension assay (PEA) to simultaneously measure 1,104 proteins in 120 longitudinal serum samples (30 ovarian cancer patients). We identified 23 candidate personalized markers (plus CA125 and FDA-approved marker HE4), in which personalized combinations was informative of recurrence in more patients (92%) compared to clinical CA125 (68%) and HE4 (32%) alone. For our ensuing validation study, we used PEAs to concurrently measure 644 proteins (includes 21 previously identified candidates plus CA125 and HE4) in 234 independent, longitudinal serum samples (39 ovarian cancer patients). The 21 candidates were each informative of recurrence in 3-35% of patients. Patient-centric analysis of all 644 proteins generated a refined panel of 33 personalized tumour markers, which includes 18 validated candidates from our discovery study. Along with HE4, the 34-marker panel offered higher sensitivity (91%) by identifying personalized marker signatures of recurrence compared to clinical CA125 (59%) and HE4 (26%) alone. Our findings show that personalized tumour markers may offer the best lens into the rich heterogeneity of ovarian tumours compared to a single marker alone. Developing a panel of personalized markers for tracking custom signatures of tumour burden in each patient may offer excellent sensitivity for detecting recurrence early and aid in prompt clinical referral to imaging and subsequent treatment interventions.Ph.D
Integrating High-throughput Technologies for the Identification and Validation of Novel Ovarian Cancer Biomarkers
No full textOvarian cancer is the most lethal gynaecological malignancy in North America. Although survival rates are high when the disease is diagnosed at an early stage, this decreases significantly in late-stage diagnoses. The majority of cases are of epithelial origin, which can be classified into four major subtypes: serous, mucinous, endometrioid and clear cell carcinoma. Unfortunately, the clinically-approved markers for ovarian cancer - CA125 and HE4 alone - perform poorly for the mucinous, endometrioid and clear cell subtypes and thus, diagnosis of these subtypes remains a significant challenge. To this end, an integrated approach to ovarian cancer biomarker discovery was developed in this study through combining proteomics with other high-throughput platforms. Using proteomic analyses of ascites fluid from women with ovarian cancer and conditioned media of ovarian cancer cell lines, 15 high-priority candidates were identified as putative novel biomarkers using said integrated approach. Serum validation revealed two markers - folate receptor 1 and kallikrein 6 - to have comparable diagnostic ability to the clinically-approved markers, albeit with similar limitations in their ability to detect patients of the non-serous histotypes as well. Fortunately, the validation of the two in-house markers served as proof-of-principle of our integrated approach to biomarker discovery. As a result, the approach was employed on non-serous ovarian cancer tissues to identify novel markers specific to the mucinous, endometrioid and clear cell subtypes of ovarian cancer. Over 9000 unique proteins were identified in this exercise and with the use of an unbiased filtering algorithm based on transcriptomics and bioinformatics, a list of high-priority candidates for each subtype was generated. Several of the high-priority candidates have shown strong biological and molecular relevance to their respective subtypes, demonstrating the robustness and utility of the integrated approach. Future studies will need to investigate these candidates in independent serum cohorts to truly assess for their ability to diagnose their respective subtypes.Ph.D.2017-11-30 00:00:0
Rapid determination of tacrolimus and sirolimus in whole human blood by direct coupling of solid-phase microextraction to mass spectrometry via microfluidic open interface
Get PDFImmunosuppressive drugs are administered to decrease immune system activity (e.g. of patients undergoing solid organ transplant). Concentrations of immunosuppressive drugs (ISDs) in circulating blood must be closely monitored during the period of immunosuppression therapy due to adverse effects that take place when concentration levels fall outside of the very narrow therapeutic concentration range of these drugs. This study presents the rapid determination of four relevant immunosuppressive drugs (tacrolimus, sirolimus, everolimus, and cyclosporine A) in whole human blood by directly coupling solid-phase microextraction to mass spectrometry via the microfluidic open interface (Bio-SPME-MOI-MS/MS). The BioSPME-MOI-MS/MS method offers ≤ 10% imprecision of in-house prepared quality controls over a 10-day period, ≤ 10% imprecision of ClinCal® Recipe calibrators over a three-day period, and single total turnaround time of ∼ 60 min (4.5 min for high throughput). The limits of quantification were determined to be 0.8 ng mL−1 for tacrolimus, 0.7 ng mL−1 sirolimus, 1.0 ng mL−1 for everolimus, and 0.8 ng mL−1 for cyclosporine. The limits of detection were determined to be 0.3 ng mL−1 for tacrolimus, 0.2 ng mL−1 for sirolimus, 0.3 ng mL−1 for everolimus, and 0.3 ng mL−1 for cyclosporine A. The R2 values for all analytes were above 0.9992 with linear dynamic range from 1.0 mL−1 to 50.0 ng mL−1 for tacrolimus, sirolimus, and everolimus while from 2.5 ng mL−1 to 500.0 ng mL−1 for cyclosporine A. To further evaluate the performance of the present method, 95 residual whole blood samples of tacrolimus and sirolimus from patients undergoing immunosuppression therapy were used to compare the Bio-SPME-MOI-MS/MS method against a clinically validated reference method based on chemiluminescent microparticle immunoassay, showing acceptable results. Our results demonstrated that Bio-SPME-MOI-MS/MS can be considered as a suitable alternative to existing methods for the determination of immunosuppressive drugs in whole blood providing faster analysis, better selectivity and sensitivity, and a wider dynamic range than current existing approaches.Fil: Nazdrajic, Emir. University of Waterloo; CanadáFil: Tascon, Marcos. University of Waterloo; Canadá. Universidad Nacional de San Martín. Instituto de Investigación e Ingeniería Ambiental. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación e Ingeniería Ambiental; ArgentinaFil: Rickert, Daniel A.. University of Waterloo; CanadáFil: Gómez Ríos, German A.. University of Waterloo; CanadáFil: Kulasingam, Vathany. University of Toronto; Canadá. University of Toronto; CanadáFil: Pawliszyn, Janusz B.. University of Waterloo; Canad
Genomic profiling for copy number changes in plasma of ovarian cancer patients – a new era for cancer diagnostics?
No full textAbstract
A blood test that can detect human malignancy with high clinical sensitivity and specificity is highly desirable. To achieve this, a tumor marker is needed that correlates with tumor burden and that can be measured with high analytical sensitivity and specificity. Over the past decades, a number of different types of tumor markers have emerged, including proteins such as enzymes, glycoproteins, and oncofetal antigens. Besides proteins, genetic abnormalities such as mutations, amplifications, and circulating tumor DNA have served as tumor markers. Despite the diversity of such biomarkers, their acceptance and implementation into routine clinical practice requires that their use results in improvements in patient outcome. Current tumor markers used in the clinic have limited utility. As such, innovative approaches to identifying tumor markers are highly desirable and one such approach may be to look for sub-chromosomal changes in the blood of patients with ovarian cancer, as is routinely performed in prenatal screening.
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http://bmcmedicine.biomedcentral.com/articles/10.1186/s12916-016-0667-
Fascin-1 is a novel biomarker of aggressiveness in some carcinomas
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Tremendous progress has been made in recent years towards the understanding, prevention and management of malignant disease, yet cancer remains a leading cause of global mortality and morbidity. Current approaches towards combating this disease include prevention, early detection and various treatment modalities. However, even with implementation of novel therapeutic options and preventative measures, most cancers are currently diagnosed at late stages, when treatment therapies are least effective. In a recent study published in BMC Medicine, Tan et al. performed a systematic review and meta-analysis to show that fascin-1, an actin-bundling protein, is associated with increased risk of mortality and metastasis in various cancer types. Although the study examined the association of fascin-1 with mortality, time-to-disease progression, lymph node metastasis and distant metastasis in five major cancer types, the clinical implications of these findings are still unclear and many unanswered questions remain.
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http://www.biomedcentral.com/1741-7015/11/5
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