Measurement of phospholipids may improve diagnostic accuracy in ovarian cancer.

BACKGROUND: More than two-thirds of women who undergo surgery for suspected ovarian neoplasm do not have cancer. Our previous results suggest phospholipids as potential biomarkers of ovarian cancer. In this study, we measured the serum levels of multiple phospholipids among women undergoing surgery for suspected ovarian cancer to identify biomarkers that better predict whether an ovarian mass is malignant. METHODOLOGY/PRINCIPAL FINDINGS: We obtained serum samples preoperatively from women with suspected ovarian cancer enrolled through a prospective, population-based rapid ascertainment system. Samples were analyzed from all women in whom a diagnosis of epithelial ovarian cancer (EOC) was confirmed and from benign disease cases randomly selected from the remaining (non-EOC) samples. We measured biologically relevant phospholipids using liquid chromatography/electrospray ionization mass spectrometry. We applied a powerful statistical and machine learning approach, Hybrid huberized support vector machine (HH-SVM) to prioritize phospholipids to enter the biomarker models, and used cross-validation to obtain conservative estimates of classification error rates. RESULTS: The HH-SVM model using the measurements of specific combinations of phospholipids supplements clinical CA125 measurement and improves diagnostic accuracy. Specifically, the measurement of phospholipids improved sensitivity (identification of cases with preoperative CA125 levels below 35) among two types of cases in which CA125 performance is historically poor - early stage cases and those of mucinous histology. Measurement of phospholipids improved the identification of early stage cases from 65% (based on CA125) to 82%, and mucinous cases from 44% to 88%. CONCLUSIONS/SIGNIFICANCE: Levels of specific serum phospholipids differ between women with ovarian cancer and those with benign conditions. If validated by independent studies in the future, these biomarkers may serve as an adjunct at the time of clinical presentation, to distinguish between women with ovarian cancer and those with benign conditions with shared symptoms and features.

Quantitative determination of lysophosphatidic acid by LC/ESI/MS/MS employing a reversed phase HPLC column.

Lysophosphatidic acid (LPA) is a class of lipids that play multiple biological functions. Several reports show that they are potential biomarkers for diagnosing ovarian cancer. Therefore, it is necessary to accurately quantify their levels in biological samples. Here we report a high throughput LC/ESI/MS/MS (liquid chromatography electrospray tandem mass spectrometry) method employing a reversed phase C18 column to quantify LPA. In this method, a [(13)C(16)] labeled 16:0 LPA is used as the internal standard and the lipids are extracted out from biological samples using Bligh-Dyer method under highly acidic condition. The total run time is 8min. The detection limits of the assay reach fmol level and the CV% of the assay are within 10%. Using this method, we quantify the levels of six LPA species (16:0, 18:2, 18:1, 18:0, 20:4, and 22:6 LPA) in plasma samples. We find that some unknown compounds present in plasma can interfere with the quantification of LPA if they are not well separated from LPA. These unknown compounds are more hydrophobic than LPA and can be removed by thin-layer chromatography (TLC). We also find that the levels of LPA species in human plasma generally follow the order: 18:2 LPA>16:0 LPA, 20:4 LPA>18:1 LPA, 22:6 LPA, and 18:0 LPA.

Quantitative determination of cyclic phosphatidic acid in human serum by LC/ESI/MS/MS.

An LC/ESI/MS/MS method for cyclic phosphatidic acid (cPA) quantification in serum is established in the present report. The limit of quantitation of the assay reaches low nanomolar level in human serum and the CV% are within 10%. Using this method, we successfully quantify the levels of two cPA species, 16:0 and 18:1, in human serum. We find that the concentrations of 16:0 cPA in the serum of normal subjects and post-surgery ovarian cancer patients are significantly higher than its corresponding concentration in pre-surgery ovarian cancer patients, supporting the observation that cPA has anti-cancer activity. Another discovery is that the addition of strong acids (such as hydrochloric acid) in human serum may lead to the production of artificial cPA. Therefore, strong acids should be avoided in the extraction of cPA present in a complex matrix. Based on this observation, a new lipid extraction method was developed and used to extract cPA. The extraction recovery is close to 80%, guaranteeing an accurate quantification of cPA by LC/ESI/MS/MS can be performed.