Development and clinical testing of individual immunoassays for the quantification of serum glycoproteins to diagnose prostate cancer

Prostate Cancer (PCa) diagnosis is currently hampered by the high false-positive rate of PSA evaluations, which consequently may lead to overtreatment. Non-invasive methods with increased specificity and sensitivity are needed to improve diagnosis of significant PCa. We developed and technically validated four individual immunoassays for cathepsin D (CTSD), intercellular adhesion molecule 1 (ICAM1), olfactomedin 4 (OLFM4), and thrombospondin 1 (THBS1). These glycoproteins, previously identified by mass spectrometry using a Pten mouse model, were measured in clinical serum samples for testing the capability of discriminating PCa positive and negative samples. The development yielded 4 individual immunoassays with inter and intra-variability (CV) <15% and linearity on dilution of the analytes. In serum, ex vivo protein stability (<15% loss of analyte) was achieved for a duration of at least 24 hours at room temperature and 2 days at 4°C. The measurement of 359 serum samples from PCa positive (n = 167) and negative (n = 192) patients with elevated PSA (2-10 ng/ml) revealed a significantly improved accuracy (P <0.001) when two of the glycoproteins (CTSD and THBS1) were combined with %fPSA and age (AUC = 0.8109; P <0.0001; 95% CI = 0.7673-0.8545). Conclusively, the use of CTSD and THBS1 together with commonly used parameters for PCa diagnosis such as %fPSA and age has the potential to improve the diagnosis of PCa

Stability experiments.

<p>Stability of CTSD, ICAM1, OLFM4, and THBS1 in serum stored at room temperature (RT), 4°C or 1 up to 3 freeze and thaw (F/T) cycles, respectively. Shown are the mean (SD; error bars) measured protein concentrations (as a percentage of the original values) in 5 different samples.</p

Measuring range and precision.

<p>Measuring range and precision.</p

Assay calibration curves.

<p>Representative calibration curves for the CTSD, ICAM1, OLFM4, and THBS1 assays.</p

Univariate analysis of individual parameters.

<p>Box plots show the concentration of PSA, %fPSA, CTSD, ICAM1, OLFM4, and THBS1 in PCa positive and negative samples.</p

Patient characteristics overview.

<p>Patient characteristics overview.</p

Receiver operating characteristics (ROC) of the individual analytes.

<p>Receiver operating characteristics (ROC) of the individual analytes.</p

SOS1 is the second most common Noonan gene but plays no major role in cardio‐facio‐cutaneous syndrome

Background: Heterozygous gain-of-function mutations in various genes encoding proteins of the Ras-MAPK signalling cascade have been identified as the genetic basis of Noonan syndrome (NS) and cardio-facio-cutaneous syndrome (CFCS). Mutations of SOS1, the gene encoding a guanine nucleotide exchange factor for Ras, have been the most recent discoveries in patients with NS, but this gene has not been studied in patients with CFCS. Methods and results: We investigated SOS1 in a large cohort of patients with disorders of the NS–CFCS spectrum, who had previously tested negative for mutations in PTPN11, KRAS, BRAF, MEK1 and MEK2. Missense mutations of SOS1 were discovered in 28% of patients with NS. In contrast, none of the patients classified as having CFCS was found to carry a pathogenic sequence change in this gene. Conclusion: We have confirmed SOS1 as the second major gene for NS. Patients carrying mutations in this gene have a distinctive phenotype with frequent ectodermal anomalies such as keratosis pilaris and curly hair. However, the clinical picture associated with SOS1 mutations is different from that of CFCS. These findings corroborate that, despite being caused by gain-of-function mutations in molecules belonging to the same pathway, NS and CFCS scarcely overlap genotypically