MicroRNAs have emerged as powerful regulators of many biological processes, and their expression in many cancer tissues has been shown to correlate with clinical parameters such as cancer type and prognosis. Present in a variety of biological fluids, microRNAs have been described as a ‘gold mine’ of potential noninvasive biomarkers. Release of microRNA content of blood cells upon hemolysis dramatically alters the microRNA profile in blood, potentially affecting levels of a significant number of proposed biomarker microRNAs and, consequently, accuracy of serum or plasma-based tests. Several methods to detect low levels of hemolysis have been proposed; however, a direct comparison assessing their sensitivities is currently lacking. In this study, we evaluated the sensitivities of four methods to detect hemolysis in serum (listed in the order of sensitivity): measurement of hemoglobin using a Coulter® AcT diff™ Analyzer, visual inspection, the absorbance of hemoglobin measured by spectrophotometry at 414 nm and the ratio of red blood cell-enriched miR-451a to the reference microRNA miR-23a-3p. The miR ratio detected hemolysis down to approximately 0.001%, whereas the Coulter® AcT diff™ Analyzer was unable to detect hemolysis lower than 1%. The spectrophotometric method could detect down to 0.004% hemolysis, and correlated with the miR ratio. Analysis of hemolysis in a cohort of 86 serum samples from cancer patients and healthy controls showed that 31 of 86 (36%) were predicted by the miR ratio to be hemolyzed, whereas only 8 of these samples (9%) showed visible pink discoloration. Using receiver operator characteristic (ROC) analyses, we identified absorbance cutoffs of 0.072 and 0.3 that could identify samples with low and high levels of hemolysis, respectively. Overall, this study will assist researchers in the selection of appropriate methodologies to test for hemolysis in serum samples prior to quantifying expression of microRNAs.

The tumour suppressor p53 is mutated in cancer, including over 96% of high-grade serous ovarian cancer (HGSOC). Mutations cause loss of wild-type p53 function due to either gain of abnormal function of mutant p53 (mutp53), or absent to low mutp53. Massively parallel sequencing (MPS) enables increased accuracy of detection of somatic variants in heterogeneous tumours. We used MPS and immunohistochemistry (IHC) to characterise HGSOCs for TP53 mutation and p53 expression. TP53 mutation was identified in 94% (68/72) of HGSOCs, 62% of which were missense. Missense mutations demonstrated high p53 by IHC, as did 35% (9/26) of non-missense mutations. Low p53 was seen by IHC in 62% of HGSOC associated with non-missense mutations. Most wild-type TP53 tumours (75%, 6/8) displayed intermediate p53 levels. The overall sensitivity of detecting a TP53 mutation based on classification as ‘Low’, ‘Intermediate’ or ‘High’ for p53 IHC was 99%, with a specificity of 75%. We suggest p53 IHC can be used as a surrogate marker of TP53 mutation in HGSOC; however, this will result in misclassification of a proportion of TP53 wild-type and mutant tumours. Therapeutic targeting of mutp53 will require knowledge of both TP53 mutations and mutp53 expression.

Background: There is a critical need for improved diagnostic markers for high grade serous epithelial ovarian cancer (SEOC). MicroRNAs are stable in the circulation and may have utility as biomarkers of malignancy. We investigated whether levels of serum microRNA could discriminate women with high-grade SEOC from age matched healthy volunteers. Methods: To identify microRNA of interest, microRNA expression profiling was performed on 4 SEOC cell lines and normal human ovarian surface epithelial cells. Total RNA was extracted from 500 μL aliquots of serum collected from patients with SEOC (n = 28) and age-matched healthy donors (n = 28). Serum microRNA levels were assessed by quantitative RT-PCR following preamplification. Results: microRNA (miR)-182, miR-200a, miR-200b and miR-200c were highly overexpressed in the SEOC cell lines relative to normal human ovarian surface epithelial cells and were assessed in RNA extracted from serum as candidate biomarkers. miR-103, miR-92a and miR -638 had relatively invariant expression across all ovarian cell lines, and with small-nucleolar C/D box 48 (RNU48) were assessed in RNA extracted from serum as candidate endogenous normalizers. No correlation between serum levels and age were observed (age range 30-79 years) for any of these microRNA or RNU48. Individually, miR-200a, miR-200b and miR-200c normalized to serum volume and miR-103 were significantly higher in serum of the SEOC cohort (P < 0.05; 0.05; 0.0005 respectively) and in combination, miR-200b + miR-200c normalized to serum volume and miR-103 was the best predictive classifier of SEOC (ROC-AUC = 0.784). This predictive model (miR-200b + miR-200c) was further confirmed by leave one out cross validation (AUC = 0.784). Conclusions: We identified serum microRNAs able to discriminate patients with high grade SEOC from age-matched healthy controls. The addition of these microRNAs to current testing regimes may improve diagnosis for women with SEOC.

Ubiquitination has traditionally been viewed in the context of polyubiquitination that is essential for marking proteins for degradation via the proteasome. Recent discoveries have shed light on key cellular roles for monoubiquitination, including as a post-translational modification (PTM) of histones such as histone H2B. Monoubiquitination plays a significant role as one of the largest histone PTMs, alongside smaller, better-studied modifications such as methylation, acetylation and phosphorylation. Monoubiquitination of histone H2B at lysine 120 (H2Bub1) has been shown to have key roles in transcription, the DNA damage response and stem cell differentiation. The H2Bub1 enzymatic cascade involves E3 RING finger ubiquitin ligases, with the main E3 generally accepted to be the RNF20–RNF40 complex, and deubiquitinases including ubiquitin-specific protease 7 (USP7), USP22 and USP44. H2Bub1 has been shown to physically disrupt chromatin strands, fostering a more open chromatin structure accessible to transcription factors and DNA repair proteins. It also acts as a recruiting signal, actively attracting proteins with roles in transcription and DNA damage. H2Bub1 also appears to play central roles in histone cross-talk, influencing methylation events on histone H3, including H3K4 and H3K79. Most significantly, global levels of H2Bub1 are low to absent in advanced cancers including breast, colorectal, lung and parathyroid, marking H2Bub1 and the enzymes that regulate it as key molecules of interest as possible new therapeutic targets for the treatment of cancer. This review offers an overview of current knowledge regarding H2Bub1 and highlights links between dysregulation of H2Bub1-associated enzymes, stem cells and malignancy.

The chemokine CXCL1 is elevated in plasma and ascites from patients with ovarian cancer. We have previously shown that CXCL1 is a marker of phosphatidylinositol 3-kinase signalling in epithelial ovarian cancer (EOC) cell lines, a pathway that is commonly activated in ovarian tumours. To investigate whether CXCL1 also has functional significance in ovarian cancer, this chemokine was either down-regulated using siRNAs or overexpressed by transfection of CXCL1 into the EOC cell lines SKOV3 and OVCAR-3 and proliferation assessed over 7 days. Overexpression of CXCL1 increased proliferation of ovarian cancer cells over 7 days, while down-regulation was inhibitory. Treatment of cells with recombinant CXCL1 induced epidermal growth factor receptor (EGFR) phosphorylation at Y1068, indicating crosstalk between the CXCL1 G-protein-coupled receptor CXCR2 and the EGFR. CXCL1-induced proliferation was also decreased by inhibition of EGFR kinase activity and was dependent on extracellular matrix metalloproteinase-mediated release of heparin-binding EGF (HB-EGF). Involvement of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase 1/2 (ERK1/2) signalling was also evident since inhibition of both Ras and MEK activity decreased CXCL1-induced proliferation. CXCL1-induced ERK1/2 phosphorylation was inhibited by the MEK1 inhibitor PD98059; however, EGFR phosphorylation was unaffected, indicating that CXCL1 activation of MAPK signalling is downstream of the EGFR. Taken together, these data show that CXCL1 functions through CXCR2 to transactivate the EGFR by proteolytic cleavage of HB-EGF, leading to activation of MAPK signalling and increased proliferation of EOC cells.

Introduction: Serum profiling using proteomic techniques has great potential to detect biomarkers that might improve diagnosis and predict outcome for breast cancer patients (BC). This study used surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry (MS) to identify differentially expressed proteins in sera from BC and healthy volunteers (HV), with the goal of developing a new prognostic biomarker panel. Methods: Training set serum samples from 99 BC and 51 HV subjects were applied to four adsorptive chip surfaces (anion-exchange, cation-exchange, hydrophobic, and metal affinity) and analyzed by time-of-flight MS. For validation, 100 independent BC serum samples and 70 HV samples were analyzed similarly. Cluster analysis of protein spectra was performed to identify protein patterns related to BC and HV groups. Univariate and multivariate statistical analyses were used to develop a protein panel to distinguish breast cancer sera from healthy sera, and its prognostic potential was evaluated. Results: From 51 protein peaks that were significantly up- or downregulated in BC patients by univariate analysis, binary logistic regression yielded five protein peaks that together classified BC and HV with a receiver operating characteristic (ROC) area-under-the-curve value of 0.961. Validation on an independent patient cohort confirmed the five-protein parameter (ROC value 0.939). The five-protein parameter showed positive association with large tumor size (P = 0.018) and lymph node involvement (P = 0.016). By matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS, immunoprecipitation and western blotting the proteins were identified as a fragment of apolipoprotein H (ApoH), ApoCI, complement C3a, transthyretin, and ApoAI. Kaplan-Meier analysis on 181 subjects after median follow-up of >5 years demonstrated that the panel significantly predicted disease-free survival (P = 0.005), its efficacy apparently greater in women with estrogen receptor (ER)-negative tumors (n = 50, P = 0.003) compared to ER-positive (n = 131, P = 0.161), although the influence of ER status needs to be confirmed after longer follow-up. Conclusions: Protein mass profiling by MS has revealed five serum proteins which, in combination, can distinguish between serum from women with breast cancer and healthy control subjects with high sensitivity and specificity. The five-protein panel significantly predicts recurrence-free survival in women with ER-negative tumors and may have value in the management of these patients.