BackgroundSerum protein profiling seems promising for early detection of breast cancer. However, the approach is also criticized, partly because of difficulties in validating discriminatory proteins. This study's aim is to validate three proteins previously reported to be discriminative between breast cancer cases and healthy controls. These proteins had been identified as a fragment of inter-alpha trypsin inhibitor H4 (4.3 kDa), C-terminal-truncated form of C3a des arginine anaphylatoxin (8.1 kDa) and C3a des arginine anaphylatoxin (8.9 kDa).MethodsSerum protein profiles of 48 breast cancer patients and 48 healthy controls were analyzed with surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS). Differences in protein intensity between breast cancer cases and controls were measured with the Mann-Whitney U test and adjusted for confounding in a multivariate logistic regression model.ResultsFour peaks, with mass-to-charge ratio (m/z) 4276, 4292, 8129 and 8941, were found that were assumed to represent the previously reported proteins. M/z 4276 and 4292 were statistically significantly decreased in breast cancer cases compared to healthy controls (p < 0.001). M/z 8941 was decreased in breast cancer cases (p < 0.001) and m/z 8129 was not related with breast cancer (p = 0.87). Adjustment for sample preparation day, sample storage duration and age did not substantially alter results.ConclusionM/z 4276 and 4292 both represented the previously reported 4.3 kDa protein and were both decreased in breast cancer patients, which is in accordance with the results of most previous studies. M/z 8129 was in contrast with previous studies not related with breast cancer. Remarkably, m/z 8941 was decreased in breast cancer cases whereas in previous studies it was increased. Differences in patient populations and pre-analytical sample handling could have contributed to discrepancies. Further research is needed before we can conclude on the relevance of these proteins as breast cancer biomarkers.

Many proteins have been proposed as potential biomarkers for breast cancer. Yet, validation of their discriminative value using quantitative methods has scarcely been performed. In this study, we investigated the discriminative value of six peptides that were previously proposed to be generated by breast cancer specific exoproteases: bradykinin, des-Arg(9)-bradykinin, Hyp(3)-bradykinin, and fragments of fibrinogen alpha-chain (Fib-alpha ([605-629])), complement component 4a (C4a ([1337-1350])), and interalpha trypsin inhibitor heavy chain 4 (ITIH4 ([666-687])). Their absolute serum concentrations were measured with a completely validated liquid chromatography-tandem mass spectrometric assay (LC-MS/MS) and compared between 62 newly diagnosed breast cancer patients and 62 controls matched for age and sample storage duration. Both ITIH4 ([666-687]) and des-Arg(9)-bradykinin showed statistically significantly higher median concentrations in breast cancer samples than in matched control samples. Additionally, we analyzed serum samples collected after surgical removal of the tumor, in which median ITIH4 ([666-687]) and des-Arg(9)-bradykinin concentrations were significantly decreased and not statistically significantly different from concentrations in the controls anymore. In a combined analysis, ITIH4 (666-687]) and des-Arg(9)-bradykinin independently contributed to the discrimination between cases and controls. In this study, we confirmed that the exoprotease breakdown peptides, ITIH4 (666-687]) and des-Arg(9)-bradykinin, differed between breast cancer cases and controls, supporting the potential of degradome markers for the diagnosis of breast cancer.

The obtained results particularly suggest potential for these ITIH(4)-derived peptides in the follow-up of breast cancer after surgery.

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