Serum Biomarker Signature-Based Liquid Biopsy for Diagnosis of Early-Stage Pancreatic Cancer.

PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis, with a 5-year survival of < 10% because of diffuse symptoms leading to late-stage diagnosis. That survival could increase significantly if localized tumors could be detected early. Therefore, we used multiparametric analysis of blood samples to obtain a novel biomarker signature of early-stage PDAC. The signature was derived from a large patient cohort, including patients with well-defined early-stage (I and II) PDAC. This biomarker signature was validated subsequently in an independent patient cohort. PATIENTS AND METHODS: The biomarker signature was derived from a case-control study, using a Scandinavian cohort, consisting of 16 patients with stage I, 132 patients with stage II, 65 patients with stage III, and 230 patients with stage IV PDAC, and 888 controls. This signature was validated subsequently in an independent case-control cohort in the United States with 15 patients with stage I, 75 patients with stage II, 15 patients with stage III, and 38 patients with stage IV PDAC, and 219 controls. An antibody microarray platform was used to identify the serum biomarker signature associated with early-stage PDAC. RESULTS: Using the Scandinavian case-control study, a biomarker signature was created, discriminating samples derived from patients with stage I and II from those from controls with a receiver operating characteristic area under the curve value of 0.96. This signature, consisting of 29 biomarkers, was then validated in an independent case-control study in the United States. The biomarker signature could discriminate patients with stage I and II PDAC from controls in this independent patient cohort with a receiver operating characteristic area under the curve value of 0.96. CONCLUSION: This serum biomarker signature might represent a tenable approach to detecting early-stage, localized PDAC if these findings are supported by a prospective validation study.

Cruciferous vegetable supplementation in a controlled diet study alters the serum peptidome in a GSTM1-genotype dependent manner.

BACKGROUND: Cruciferous vegetable intake is inversely associated with the risk of several cancers. Isothiocyanates (ITC) are hypothesized to be the major bioactive constituents contributing to these cancer-preventive effects. The polymorphic glutathione-S-transferase (GST) gene family encodes several enzymes which catalyze ITC degradation in vivo. METHODS: We utilized high throughput proteomics methods to examine how human serum peptides (the "peptidome") change in response to cruciferous vegetable feeding in individuals of different GSTM1 genotypes. In two randomized, crossover, controlled feeding studies (EAT and 2EAT) participants consumed a fruit- and vegetable-free basal diet and the basal diet supplemented with cruciferous vegetables. Serum samples collected at the end of the feeding period were fractionated and matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry spectra were obtained. Peak identification/alignment computer algorithms and mixed effects models were used to analyze the data. RESULTS: After analysis of spectra from EAT participants, 24 distinct peaks showed statistically significant differences associated with cruciferous vegetable intake. Twenty of these peaks were driven by their GSTM1 genotype (i.e., GSTM1+ or GSTM1- null). When data from EAT and 2EAT participants were compared by joint processing of spectra to align a common set, 6 peaks showed consistent changes in both studies in a genotype-dependent manner. The peaks at 6700 m/z and 9565 m/z were identified as an isoform of transthyretin (TTR) and a fragment of zinc α2-glycoprotein (ZAG), respectively. CONCLUSIONS: Cruciferous vegetable intake in GSTM1+ individuals led to changes in circulating levels of several peptides/proteins, including TTR and a fragment of ZAG. TTR is a known marker of nutritional status and ZAG is an adipokine that plays a role in lipid mobilization. The results of this study present evidence that the GSTM1-genotype modulates the physiological response to cruciferous vegetable intake.

Evaluation of matrix-assisted laser desorption/ionization-time of flight mass spectrometry proteomic profiling: identification of alpha 2-HS glycoprotein B-chain as a biomarker of diet.

Biomarkers have the potential to impact a wide range of public health concerns, including early detection of diseases, drug discovery, and improved accuracy of monitoring effects of interventions. Given new technological developments, broad-based screening approaches will likely advance biomarker discovery at an accelerated pace. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) allows for the elucidation of individual protein masses from a complex mixture with high throughput. We have developed a method for identifying serum biomarkers using MALDI-TOF and statistical analysis. However, before applying this approach to screening of complex diseases, we evaluated the approach in a controlled dietary intervention study. In this study, MALDI-TOF spectra were generated using samples from a randomized controlled trial. During separate feeding periods, 38 participants ate a basal diet devoid of fruits and vegetables and a basal diet supplemented with cruciferous (broccoli) family vegetables. Serum samples were obtained at the end of each 7-day feeding period and treated to remove large, abundant proteins. MALDI-TOF spectra were analyzed using peak picking algorithms and logistic regression models. Our bioinformatics methods identified two significant peaks at m/z values of 2740 and 1847 that could classify participants based on diet (basal vs. cruciferous) with 76% accuracy. The 2740 m/z peak was identified as the B-chain of alpha 2-HS glycoprotein, a serum protein previously found to vary with diet and be involved in insulin resistance and immune function.

Impact of freeze-thaw cycles and storage time on plasma samples used in mass spectrometry based biomarker discovery projects.

Mass spectrometry approaches to biomarker discovery in human fluids have received a great deal of attention in recent years. While mass spectrometry instrumentation and analysis approaches have been widely investigated, little attention has been paid to how sample handling can impact the plasma proteome and therefore influence biomarker discovery. We have investigated the effects of two main aspects of sample handling on MALDI-TOF data: repeated freeze-thaw cycles and the effects of long-term storage of plasma at -70° C. Repeated freeze-thaw cycles resulted in a trend towards increasing changes in peak intensity, particularly after two thaws. However, a 4-year difference in long-term storage appears to have minimal effect on protein in plasma as no differences in peak number, mass distribution, or coefficient of variation were found between samples. Therefore, limiting freeze/thaw cycles seems more important to maintaining the integrity of the plasma proteome than degradation caused by long-term storage at -70° C.