An integrated approach to protein discovery and detection from complex biofluids

Ovarian cancer, a leading cause of cancer related deaths among women, has been notoriously difficult to routinely screen for and diagnose early. Researchers and clinicians continue to seek routinely usable, non-invasive, screening methods as early detection significantly improves survival. Biomarker screening is ideal; however, currently available ovarian cancer biomarkers lack desirable sensitivity and specificity. Furthermore, the most fatal forms, high grade serous cancers often originate in the fallopian tube; therefore, sampling from the vaginal environment provides more proximal sources for tumor detection. To address these shortcomings and leverage proximal sampling, we developed an untargeted mass spectrometry microprotein profiling method and identified a signature of cystatin A, validated this protein in an animal model, and sought to overcome the limits of detection inherent to mass spectrometry by demonstrating that cystatin A is present at 100 pM concentrations using a label-free microtoroid resonator. The findings highlight the potential utility for early-stage detection where cystatin A levels would be low.

Gonadotropin Stimulation Has Only a Limited Effect on the Concentration of Follicular Fluid Signalling Proteins: An Antibody Array Analysis

Objective. The follicular fluid (FF) plays an essential role in the physiology of the follicle and the oocyte. Gonadotropin stimulation affects the FF steroid hormone and anti-Mullerian hormone (AMH) concentrations, which has been suggested to be the reason for lower oocyte competence in conventional gonadotropin stimulated in vitro fertilisation (cIVF) compared to natural cycle IVF (NC-IVF). To analyse the effect of gonadotropin stimulation on a broad spectrum of signalling proteins, we ran proteomic antibody arrays on FF of women undergoing both treatments NC-IVF and cIVF. Method. Twenty women underwent one NC-IVF and one cIVF treatment cycle. Follicular fluids of the first aspirated follicle were compared between the two groups using a protein microarray which included antibodies against 224 proteins related to cell signalling and reference proteins. Each of the 40 albumin-stripped, matched-pair samples was labelled in the reverse-dye (Cy3/Cy5) procedure before undergoing array hybridisation. Signal analysis was performed using normalisation algorithms in dedicated software. Five proteins yielding a value of P < 0.05 in the array experiment (Cystatin A, Caspase-3, GAD65/67, ERK-1, and ERK-2) were then submitted to quantitative determination by ELISA in the same follicular fluids. Results. Array analysis yielded only a small number of differentially expressed signalling markers by unadjusted P values. Adjustment as a consequence of multiple determinations resulted in the absence of any significant differential marker expression on the array. Five unadjusted differentially expressed proteins were quantified immunometrically with antibodies from different sources. Follicular fluid concentrations of Cystatin A and MAP kinase ERK-1 concentrations were significantly higher in the cIVF than in the NC-IVF follicles, while GAD-2 (GAD65/67) did not differ. The assays for Caspase-3 and MAP kinase ERK-2 did not have the required sensitivities. Conclusion. In contrast to FF steroid hormones and AMH, FF concentrations of signalling proteins are not or only marginally altered by gonadotropin stimulation.

Identification of CD37, cystatin A, and IL-23A gene expression in association with brain metastasis: analysis of a prospective trial

Purpose/Objectives: We aimed to assess the predictive value of a lung cancer gene panel for the development of brain metastases. Materials/Methods: Between 2011 and 2015, 102 patients with lung cancer were prospectively enrolled in a clinical trial in which a diagnostic fine-needle aspirate was obtained. Gene expression was conducted on all samples that rendered a diagnosis of non-small cell lung cancer (NSCLC). Subsequent retrospective analysis of brain metastases-related outcomes was performed by reviewing patient electronic medical records. A competing risk multivariable regression was performed to estimate the adjusted hazard ratio for the development of brain metastases and non-brain metastases from NSCLC. Results: A total of 49 of 102 patients had died by the last follow-up. Median time of follow-up was 13 months (range 0.23–67 months). A total of 17 patients developed brain metastases. Median survival time after diagnosis of brain metastases was 3.58 months (95% confidence interval (CI) 2.17, not available). A total of 30 patients developed metastases without any evidence of brain metastases until the time of death or last follow-up. Competing risk analysis identified three genes that were downregulated differentially in the patients with brain metastases versus non-brain metastatic disease: CD37 (0.017), cystatin A (0.022), and IL-23A (0.027). Other factors associated with brain metastases include: stage T ( P ⩽ 8.3e-6) and stage N ( P= 6.8e-4). Conclusions: We have identified three genes, CD37, cystatin A, and IL-23A, for which downregulation of gene expression was associated with a greater propensity for developing brain metastases. Validation of these biomarkers could have implications on surveillance patterns in patients with brain metastases from NSCLC.