To date, few studies have addressed the temporal reliability of chemokines, cancer antigens, growth factors, apoptotic factors, and adipokines in healthy subjects. Our results are consistent with previous studies of CA 15-3 [15
], MCP-1 [16
], and RANTES [17
]. To our knowledge, our group is the first to evaluate the temporal reliability of the majority of these biomarkers in healthy individuals, as, other than CA 15-3, CA 125, MCP-1, and RANTES, few markers have been explored in previous research of temporal reliability.
CA 125 has been used extensively for the diagnosis and follow-up of ovarian cancer patients [18
]. A previous study evaluating CA 125 in healthy, menopausal women, using radioimmunoassay suggested that single, low CA 125 values are reliable indicators of a woman’s true CA 125 value [19
]. Using the multiplexing method, we confirmed that CA 125 is a reliable marker.
Resistin, a recently discovered adipokine, is purportedly involved in metabolic and inflammatory processes in humans and may be an important marker with which to assess disease risk in large-scale epidemiological studies. In our study, resistin was one of the most reliable markers, which confirmed the results of a recent study using ELISA. In that study, individual blood resistin concentrations did not significantly change over a period of one year, and showed a high degree of reliability [20
In general, very limited number of studies have evaluated longitudinal changes of these biological markers in healthy individuals [9
]. The majority of existing studies relied on correlations and did not report the variance components or ICCs, which provide superior assessment of reliability. Additionally, most of the existing studies on biomarker reliability to date, do not assess markers in healthy participants, evaluating only biomarker changes in patients with various benign and malignant conditions. In this study, serum levels of most of the biomarkers were similar to those measured by the same xMAP™ method in other studies [5
], and those measured by ELISA in healthy populations. The differences in population characteristics (age, gender, etc.), assay sensitivity and specificity, standards used in the assays [21
], or sample collection, processing, storage and assay performance [22
], may contribute to the observed differences in biomarker concentrations in healthy subjects. Therefore, standardization of procedures needs to be done before there can be any direct comparison between studies.
Previous studies have suggested that the circulating levels of serum biomarkers can be affected by a wide range of factors, including age, gender, race, blood pressure, serum cholesterol, BMI, percentage body fat, visceral fat, cigarette smoking, the use of hormone replacement therapy, menopausal status, and physical exercise [23
]. Reliability studies in the area of biomarkers are complicated by the fact that “normal” levels of biomarkers may differ with age. One of the previous studies found elevated cancer antigen levels in elderly individuals without any confirmed malignancies [24
], suggesting that biomarkers may change over time due to the aging process rather than to occult pathology. Despite these factors affecting biomarker reliability, our study has demonstrated that a substantial number of biomarkers were stable over a one-two year period.
Our study had some limitations. The samples were not assayed in duplicate. However, this is common in assays using the Luminex method, which provides an average value based on 100 bead measurements. Our study population included women only, so the results may not be extrapolated to males. Future studies need to look into the reliability of biomarkers in premenopausal versus postmenopausal women in more detail, and compare the reliability of biomarkers in males and females. Despite these limitations, this is one of the first and the largest studies assessing the reliability of multiple serum markers using Luminex methodology.
In addition to addressing these limitations, in our future studies it would be important to evaluate more carefully the presence of various biological markers implicated in cancer development in the serum of healthy individuals. The presence of these biomarkers in the serum of healthy individuals is still not well understood. A good example of this concept is ErbB2, a member of the epidermal growth factor receptor family, implicated in the development of many human cancers. At this point, the presence of ErbB2 in serum samples from healthy individuals has not been explored by large epidemiological studies. Since the presence of overexpression of ErbB2 in serum of healthy individuals could be symptomatic of the development of breast cancer or cancer in general, the detection of high and reliable-over-time levels of ErbB2 in the blood of healthy individuals could be an indication for the continuation of more frequent tests to unveil the cause.
Additionally, this study resulted in novel data on the differences between biomarker expression levels in healthy, premenopausal and healthy, postmenopausal women over time. Due to a relatively small size of this cohort, only very large differences between pre- and postmenopausal women would be detectable in our study. This study detected differences between premenopausal versus postmenopausal women in 15 out 55 markers, including TTR, eotaxin, GRO-α, PAI-1(active), fractalkine, sICAM-1, sE-Selectin, tPAI-1, SCC, thrombospondin, MMP-2, MCP-1, CA-125, SAA, and resistin. These results were consistent with previous research of healthy women followed up through menopausal transition, which suggested that SAA, tPAI, and MCP-1 differ between premenopausal and postmenopausal status [25
]. Additionally, previous evidence suggests that there are differences in PAI-1, MMP-1, and MMP-2 levels between healthy, premenopausal versus postmenopausal women [26
]. Consistently with our study, Grover et al
. found that both hysterectomy and menopausal status have a clear effect on serum CA 125 levels and must be considered if serum CA 125 is to be used as a screening test [28
]. Other than SAA, MCP-1, PAI-1, MMP-1, and MMP-2, the rest of the markers in our study that were differentially expressed between postmenopausal and premenopausal women have been only rarely investigated in healthy women in relation to menopausal status. In conclusion, using the xMAP™ method we found that serum concentrations of cancer antigens: AFP, CA 15-3, CEA, CA-125, SCC, SAA; growth factors/related molecules: ErbB2, IGFBP-1; proteases and adhesion molecules: MMP-1,8,9, sE-selectin, KLK8,10, sICAM-1, sVCAM-1; chemokines: fractalkine, MCP-1,2, RANTES, MIP-1α, MIP-1β, eotaxin, GRO-α, IP-10; angiogenesis inhibitors: angiostatin and endostatin; adipokines: leptin and resistin; apoptotic factor: sFas; and other proteins: mesothelin, MPO, and PAI-1, are detectable and remain stable for up to two years in stored serum samples, suggesting that a single measurement of this markers may be sufficient for utilization in clinical and epidemiological studies.