Detection of Cervical Cancer Biomarker Patterns in Blood Plasma and Urine by Differential Scanning Calorimetry and Mass Spectrometry

Abstract

Improved methods for the accurate identification of both the presence and severity of cervical intraepithelial neoplasia (CIN) and extent of spread of invasive carcinomas of the cervix (IC) are needed. Differential scanning calorimetry (DSC) has recently been shown to detect specific changes in the thermal behavior of blood plasma proteins in several diseases. This methodology is being explored to provide a complementary approach for screening of cervical disease. The present study evaluated the utility of DSC in differentiating between healthy controls, increasing severity of CIN and early and advanced IC. Significant discrimination was apparent relative to the extent of disease with no clear effect of demographic factors such as age, ethnicity, smoking status and parity. Of most clinical relevance, there was strong differentiation of CIN from healthy controls and IC, and amongst patients with IC between FIGO Stage I and advanced cancer. The observed disease-specific changes in DSC profiles (thermograms) were hypothesized to reflect differential expression of disease biomarkers that subsequently bound to and affected the thermal behavior of the most abundant plasma proteins. The effect of interacting biomarkers can be inferred from the modulation of thermograms but cannot be directly identified by DSC. To investigate the nature of the proposed interactions, mass spectrometry (MS) analyses were employed. Quantitative assessment of the low molecular weight protein fragments of plasma and urine samples revealed a small list of peptides whose abundance was correlated with the extent of cervical disease, with the most striking plasma peptidome data supporting the interactome theory of peptide portioning to abundant plasma proteins. The combined DSC and MS approach in this study was successful in identifying unique biomarker signatures for cervical cancer and demonstrated the utility of DSC plasma profiles as a complementary diagnostic tool to evaluate cervical cancer health.

Use of Differential Scanning Calorimetry and Immunoaffinity Chromatography to Identify Disease Induced Changes in Human Blood Plasma Proteome

“…Chaires et al have shown that thermograms of plasma from diseased individuals differ significantly from thermograms of plasma from healthy individuals. 2,4 Another tool to facilitate the search for biomarkers is liquid chromatography, in particular affinity chromatography and ion-exchange chromatography. Several researchers have demonstrated that depletion of most abundant plasma proteins amplifies the contribution of least abundant plasma proteins, thus creating the opportunity to uncover and study specific proteins/peptides.…”

Evaluation of the safety and pharmacokinetic profile of the broad spectrum antiviral lectin Griffithsin.

“…The first moment temperature (T fm ), used to analyze geometric area distribution changes, was calculated as described by Garbett [231]. Additionally, excess specific heat capacities were analyzed for the following temperature points: 55, 60, 65, 67, 70, 75, and 80ºC.…”

“…For generation of rat serum thermogram profiles after spiking with PBS or GRFT, the protocol for sample preparation and dialysis described by Garbett et al was used [231]. Briefly, a standard phosphate dialysis buffer (1.7 mM KH 2 PO 4 , 8.3 mM K 2 HPO, 150 mM NaCl, 15 mM Na 3 C 6 H 5 O 7 , pH 7.5) was formulated and rat serum was dialyzed for a period of 24 hours at 4ºC to normalize buffer conditions.…”

“…These thermograms represent the weighted average of all the major serum proteins interactions [230]. Alterations in thermograms from a "normal" curve generated from healthy subjects represent changes in major serum protein interactions [231]. The interactome hypothesis holds that low molecular weight proteins or peptide binding to more abundant serum proteins can alter normal serum protein interactions and thermostability [232].…”

“…Furthermore, DSC has been proven to effectively identify changes in thermogram shapes indicative of degrees of disease state in cervical cancer [231]. Therefore, we hypothesized that DSC would be a useful additional tool in determining whether systemic GRFT administration alters endogenous serum protein interactions, which in turn could lead to identification of unseen toxicities associated with GRFT treatment.…”