Proteomic-based identification of haptoglobin-1 precursor as a novel circulating biomarker of ovarian cancer

Screening for specific biomarkers of early-stage detection of ovarian cancer is a major health priority due to the asymptomatic nature and poor survival characteristic of the disease. We utilised two-dimensional gel electrophoresis (2DE) to identify differentially expressed proteins in the serum of ovarian cancer patients that may be useful as biomarkers of this disease. In this study, 38 ovarian cancer patients at different pathological grades (grade 1 (n=6), grade 2 (n=8) and grade 3 (n=24)) were compared to a control group of eight healthy women. Serum samples were treated with a mixture of Affigel-Blue and protein A (5 : 1) for 1 h to remove high abundance protein (e.g. immunoglobulin and albumin) and were displayed using 11 cm, pH 4-7 isoelectric focusing strips for the first dimension and 10% acrylamide gel electrophoresis for the second dimension. Protein spots were visualised by SYPRO-Ruby staining, imaged by FX-imager and compared and analysed by PDQuest software. A total of 24 serum proteins were differentially expressed in grade 1 (P<0.05), 31 in grade 2 (P<0.05) and 25 in grade 3 (P<0.05) ovarian cancer patients. Six of the protein spots that were significantly upregulated in all groups of ovarian cancer patients were identified by nano-electrospray quadrupole quadrupole time-of-flight mass spectrometry (n-ESIQ(q)TOFMS) and matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOFMS) as isoforms of haptoglobin-1 precursor (HAP1), a liver glycoprotein present in human serum. Further identification of the spots at different pathological grades was confirmed by Western blotting using monoclonal antibody against a haptoglobin epitope contained within HAP1. Immunohistochemical localisation of HAP1-like activity was present in malignant ovarian epithelium and stroma but strong immunostaining was present in blood vessels, areas with myxomatous stroma and vascular spaces. No tissue localisation of HAP1-like immunoreactivity was observed in normal ovarian surface epithelium. These data highlight the need to assess circulating concentration of HAP1 in the serum of ovarian cancer patients and evaluate its potential as a biomarker in the early diagnosis of ovarian cancer.N Ahmed, G Barker, KT Oliva, P Hoffmann, C Riley, S Reeve, AI Smith, BE Kemp, MA Quinn and GE Ric

Haptoglobin phenotype is not a predictor of recurrence free survival in high-risk primary breast cancer patients

Contains fulltext : 70104tjan-heijnen.pdf (publisher's version ) (Open Access)BACKGROUND: Better breast cancer prognostication may improve selection of patients for adjuvant therapy. We conducted a retrospective follow-up study in which we investigated sera of high-risk primary breast cancer patients, to search for proteins predictive of recurrence free survival. METHODS: Two sample sets of high-risk primary breast cancer patients participating in a randomised national trial investigating the effectiveness of high-dose chemotherapy were analysed. Sera in set I (n = 63) were analysed by surface enhanced laser desorption ionisation time-of-flight mass spectrometry (SELDI-TOF MS) for biomarker finding. Initial results were validated by analysis of sample set II (n = 371), using one-dimensional gel-electrophoresis. RESULTS: In sample set I, the expression of a peak at mass-to-charge ratio 9198 (relative intensity 20), identified as haptoglobin (Hp) alpha-1 chain, was strongly associated with recurrence free survival (global Log-rank test; p = 0.0014). Haptoglobin is present in three distinct phenotypes (Hp 1-1, Hp 2-1, and Hp 2-2), of which only individuals with phenotype Hp 1-1 or Hp 2-1 express the haptoglobin alpha-1 chain. As the expression of the haptoglobin alpha-1 chain, determined by SELDI-TOF MS, corresponds to the phenotype, initial results were validated by haptoglobin phenotyping of the independent sample set II by native one-dimensional gel-electrophoresis. With the Hp 1-1 phenotype as the reference category, the univariate hazard ratio for recurrence was 0.87 (95% CI: 0.56 - 1.34, p = 0.5221) and 1.03 (95% CI: 0.65 - 1.64, p = 0.8966) for the Hp 2-1 and Hp 2-2 phenotypes, respectively, in sample set II. CONCLUSION: In contrast to our initial results, the haptoglobin phenotype was not identified as a predictor of recurrence free survival in high-risk primary breast cancer in our validation set. Our initial observation in the discovery set was probably the result of a type I error (i.e. false positive). This study illustrates the importance of validation in obtaining the true clinical applicability of a potential biomarker

Alterations in the human lung proteome with lipopolysaccharide

<p>Abstract</p> <p>Background</p> <p>Recombinant human activated protein C (rhAPC) is associated with improved survival in high-risk patients with severe sepsis; however, the effects of both lipopolysaccharide (LPS) and rhAPC on the bronchoalveolar lavage fluid (BALF) proteome are unknown.</p> <p>Methods</p> <p>Using differential in gel electrophoresis (DIGE) we identified changes in the BALF proteome from 10 healthy volunteers given intrapulmonary LPS in one lobe and saline in another lobe. Subjects were randomized to pretreatment with saline or rhAPC.</p> <p>Results</p> <p>An average of 255 protein spots were detected in each proteome. We found 31 spots corresponding to 8 proteins that displayed abundance increased or decreased at least 2-fold after LPS. Proteins that decreased after LPS included surfactant protein A, immunoglobulin J chain, fibrinogen-γ, α₁-antitrypsin, immunoglobulin, and α₂-HS-glycoprotein. Haptoglobin increased after LPS-treatment. Treatment with rhAPC was associated with a larger relative decrease in immunoglobulin J chain, fibrinogen-γ, α₁-antitrypsin, and α₂-HS-glycoprotein.</p> <p>Conclusion</p> <p>Intrapulmonary LPS was associated with specific protein changes suggesting that the lung response to LPS is more than just a loss of integrity in the alveolar epithelial barrier; however, pretreatment with rhAPC resulted in minor changes in relative BALF protein abundance consistent with its lack of affect in ALI and milder forms of sepsis.</p