Original ArticlesDiscriminating patients with early-stage pancreatic cancer or chronic pancreatitis using serum electrospray mass profiling
Introduction
Early detection of pancreatic ductal adenocarcinoma (PDAC) is an important aspect of cancer treatment because early clinical stages (I, II) are easier to cure than later stages (III, IV) [1]. There is a need for robust, accurate and non-invasive detection methodology, e.g. from blood, for the early stages of pancreatic cancer [1], [2]. Serum protein CA-19.9 is used to monitor existing pancreatic cancer but is not useful in diagnosis [3]. Multiple micro (mi) RNAs from plasma were shown to be indicators for pancreatic cancer, and mi-155 is possibly predictive for early-stage pancreatic neoplasia [4]. However there are still some discrepancies among micro RNA technologies [5], [6]. A variety of serum biomarkers in an antibody-protein microarray format had positive results detecting late-stage pancreatic cancer and chronic pancreatitis (CP) [7]. Chronic pancreatitis is a significant risk factor for the development of pancreatic cancer [8], [9]. One of the prominent mechanisms by which PDAC is hypothesized to develop, e.g., from pancreatitis to pancreatic cancer, is through cellular and genetic changes involving pancreatic intraepithelial neoplasias (PanINs) which can be found in chronic pancreatitis [10], [11].
The profiling of bodily fluids using all-liquid electrospray ionization (ESI) mass spectrometry (MS) has the potential to distinguish differences between blood/sera of disease-free individuals and individuals with pathological conditions [12], [13], [14], [15]. Serum mass profiling is useful in cancer diagnostics including pancreatic cancer, and in therapeutic development [14], [15], [16], [17]. The underlying hypothesis is that sera contain ample numbers and kinds of peptides and other biomolecules (e.g., proteins, nucleic acids, glycoconjugates, lipids), and this complexity will vary between disease states [12], [13], [14], [15]. The basis for some of this complexity involves exoprotease degradation of proteins [18] and cellular signaling mechanisms [19], and is hypothesized to reflect homeostatic as well as defense/stress mechanisms which change with physiological state [16], [17], [18], [19]. Consequently, organs/tissues shed and/or secrete varying amounts and different kinds of biomolecules into the peripheral blood in response to different physiological conditions. All-liquid ESI-MS is possibly the simplest biomarker platform available, requiring only a serum dilution and injection into the mass spectrometer. Liquid MS analyzes disease-related phenotypic profiles in sera, as opposed to indirect genotypic/nucleic acid classifications. ESI-MS serum mass profiling examines potentially all biomolecules in sera, whereas other biomarker platforms (DNA, RNA, metabolomics, and various antibody methods) focus on a single component or small groups of similar components and can require a significant amount of preparation prior to analysis. To improve specificity in disease detection, the more biomolecules analyzed at once, the greater disease discriminatory powers of the platform [17], [18]. Importantly, MS analysis meets the accuracy, robustness, and reproducibility guidelines for stringent clinical laboratory testing [20], [21], [22], [23]. Standard statistical approaches, like those used in this study, are better suited than novel algorithms [20], [23].
Previously, we utilized electrospray ionization mass spectrometry (ESI-MS) peaks to distinguish sera from early-stage ovarian, lung, and pancreatic cancer patients from healthy disease-free individuals [15], [16], [17], [24], [25], [26]. In the present study, electrospray serum mass profiling is used to distinguish early-stage PDAC patients (stages I, IIA, IIB) from healthy individuals and from patients with chronic pancreatitis. Leave one out cross validation (LOOCV) of the mass peak data and randomization of cohort sera samples is used to check for and help ameliorate “over-fitting” of the mass peak data. “Hold out” databases are formed and used to validate blinded early-stage PDAC, CP, or control serum sets. Tandem MS/MS [27] is used to help identify peptides/proteins potentially involved in PDAC/control discrimination. Such straight-forward analyses, from an accessible body fluid like serum, holds promise for aiding in the diagnosis and disease monitoring and, in the future, understanding pancreatic carcinogenesis mechanisms as well as aiding in the development and analysis of therapeutic interventions for this deadly disease.
Section snippets
Patients and clinical samples
Patient-related information concerning individuals with stage I, IIA, or IIB pancreatic cancer, chronic pancreatitis, as well as healthy control individuals, is listed in Table 1. Patient/serum samples are divided into three groups: complete databases, validation databases, and blind validation samples. Tumor pathological staging was according to the TNM staging system (tumor size, node involvement, metastasis presence) [28]. Tumor and pancreatitis pathology was determined at the Surgical
Serum mass peak profiling for distinguishing patients with early-stage pancreatic cancer
The histology in Fig. 1A–C illustrates the problem addressed, i.e., trying to distinguish, in a minimally-invasive manner, patients with early-stage pancreatic cancer (panel C, stage IIB), chronic pancreatitis (panel B), from control individuals (normal pancreas, panel A). A normal duct (ND) and Islet cells (IC), fibrotic scarring (F/S) and PanIN, or ductal adenocarcinoma (PDAC) and neuronal fibers (NF) are exhibited in panels A, B, and C respectively. Development of such a diagnostic tool
Discussion
The early detection and prevention of pancreatic cancer are of utmost importance and major clinical and research interests [1], [2], [16], [38]. The earlier this disease can be diagnosed, the earlier life-saving treatments can begin, thus increasing the survival rate of this cancer much above the present 4–5% [1], [2]. Serum mass profiling is a promising technology for identifying potential biomarkers and their patterns relevant to the diagnosis, monitoring, understanding, and treating a
Funding
This study was funded by the Oklahoma Center for the Advancement of Science and Technology, and the Department of Surgery, University of Oklahoma Health Sciences Center.
Conflict of interest
The authors declare no conflicts of interest.
Acknowledgements
Authors acknowledge financial support from the Oklahoma Center for the Advancement of Science and Technology (OCAST Grant Number AR11-001), National Center for Research Resources and the National Institute of General Medical Sciences of the National Institutes of Health (Grant Number 8P20GM103447), Initiative for Minority Students: Bridges to the Baccalaureate Degree Award No: 5-R25-GM054938-10, Oklahoma Tobacco Research Center (OTRC Award C1063804), and the University of Oklahoma Health
References (47)
- et al.
Frequency of K-ras mutations in pancreatic intraductal neoplasias associated with pancreatic ductal adenocarcinoma and chronic pancreatitis: a meta-analysis
Neoplasia
(2005) - et al.
Composition of the peptide fraction in human blood plasma: database of circulating human peptides
J. Chromatogr. B. Biomed Sci. Appl
(1999) - et al.
Serum discrimination of early-stage lung cancer patients using electrospray-ionization mass spectrometry
Lung Cancer
(2011) - et al.
Mucin 16 (cancer antigen 125) expression in human tissues and cell lines and correlation with clinical outcome in adenocarcinomas of the pancreas, esophagus, stomach, and colon
Hum. Pathol
(2012) Cancer Facts & Figures 2013
(2013)- et al.
Core signaling pathways in human pancreatic cancers revealed by global genomic analyses
Science
(2008) - et al.
Tumor markers in patients with pancreatic carcinoma
Cancer
(1996) - et al.
MicroRNAs in plasma of pancreatic ductal adenocarcinoma patients as novel blood-based biomarkers of disease
Cancer Prev. Res. (Phila.)
(2009) MicroRNA profiling: separating signal from noise
Nat. Methods
(2010)- et al.
Identification and remediation of biases in the activity of RNA ligases in small-RNA deep sequencing
Nucleic Acids Res
(2011)
Identification of serum biomarker signatures associated with pancreatic cancer
Cancer Res
Risk of pancreatic adenocarcinoma in chronic pancreatitis
Gut
Inflammation, autophagy, and obesity: common features in the pathogenesis of pancreatitis and pancreatic cancer
Gastroenterology
Proliferative activity in pancreatic intraepithelial neoplasias of chronic pancreatitis resection specimens: detection of a high-risk lesion
Neoplasma
Proteomics: a new approach to the study of disease
J. Pathol
Proteomic approaches within the NCI early detection research network for the discovery and identification of cancer biomarkers
Ann. N. Y. Acad. Sci
Biomarker identification in human pancreatic cancer sera
Pancreas
Distinguishing early-stage pancreatic cancer patients from disease-free individuals using serum profiling
Cancer Invest
Mass profiling of serum to distinguish mice with pancreatic cancer induced by a transgenic Kras mutation
Int. J. Cancer
Differential exoprotease activities confer tumor-specific serum peptidome patterns
J. Clin. Invest
DAMPs, PAMPs, and alarmins: all we need to know about danger
J. Leukoc. Biol
Evaluation of serum protein profiling by surface-enhanced laser esorption/ionization time-of- flight mass spectrometry for the detection of prostate cancer: I. Assessment of platform reproducibility
Clin. Chem
Can mass spectrometric protein profiling meet desired standards of clinical laboratory practice?
Clin. Chem
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