Cancer Letters

Cancer Letters

Volume 359, Issue 2, 10 April 2015, Pages 314-324
Cancer Letters

Original Articles
Discriminating patients with early-stage pancreatic cancer or chronic pancreatitis using serum electrospray mass profiling

https://doi.org/10.1016/j.canlet.2015.01.035Get rights and content

Highlights

  • We evaluated and distinguished patients with early-stage PDAC using serum ESI-MS profiling.

  • We evaluated and distinguished patients with chronic pancreatitis using serum ESI-MS profiling.

  • We validated blind patient samples to the appropriate patient groups.

  • We observed a cancer phenotype with elements of PDAC in early-stage PDAC/control discrimination.

Abstract

Blood tests are needed to aid in the early detection of pancreatic ductal adenocarcinoma (PDAC), and monitoring pancreatitis development into malignancy especially in high risk patients. This study exhibits efforts and progress toward developing such blood tests, using electrospray-mass spectrometry (MS) serum profiling to distinguish patients with early-stage PDAC or pancreatitis from each other and from controls. Identification of significant serum mass peak differences between these individuals was performed using t tests and “leave one out” cross validation. Serum mass peak distributions of control individuals were distinguished from those of patients with chronic pancreatitis or early-stage PDAC with P values <10−15, and patients with chronic pancreatitis were distinguished from those of patients with early-stage PDAC with a P value <10−12. Sera from 12 out of 12 patients with PDAC stages I, IIA and IIB were blindly validated from controls. Tandem MS/MS identified a cancer phenotype with elements of PDAC involved in early-stage PDAC/control discrimination. These studies indicate electrospray-MS mass profiling can detect serum changes in patients with pancreatitis or early-stage pancreatic cancer. Such technology has the potential to aid in early detection of pancreatic cancer, biomarker development, and in monitoring development of pancreatitis into PDAC.

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

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