Identification and validation of novel candidate protein biomarkers for the detection of human gastric cancer

https://doi.org/10.1016/j.bbapap.2014.01.018Get rights and content

Highlights

  • Novel protein biomarkers for Late Stage (Stages II and III) and Early Stage (Stage I) gastric cancer

  • Validating novel protein biomarkers by ELISA and MRM-HR

  • Novel isoform specific biomarkers for gastric cancer

Abstract

The timely detection of gastric cancer will contribute significantly towards effective treatment and is aided by the availability and reliability of appropriate biomarkers. A combination of several biomarkers can improve the sensitivity and specificity of cancer detection and this work reports results from a panel of 4 proteins. By combining a validated preclinical mouse model with a proteomic approach we have recently discovered novel biomarkers for the detection of gastric cancer. Here, we investigate the specificity of four of those biomarkers (afamin, clusterin, VDBP and haptoglobin) for the detection of gastric cancer using two independent methods of validation: ELISA, and a non antibody based method: Multiple Reaction Monitoring with High Resolution Mass Spectrometry (MRM-HR). All four biomarkers reliably differentiated GC from benign patient serum, and also in a small cohort of 11 early stage cases. We also present a novel isoform specific biomarker alpha-1-antitrypsin (A1AT) that was identified using a mouse model for gastric cancer. This isoform is distinct in charge and mobility in a pH gradient and was validated using human samples by isoelectric focussing and Western-blot (IEF-WB). This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.

Introduction

Gastric cancer (GC) is the fourth most common cancer in the world and the second leading cause of death due to cancer, reflecting the overall 5 year survival rate (5YSR) of 24% [1]. This poor outcome can be attributed to an extended asymptomatic period associated with this cancer, and difficulty in the detection of early stage gastric adenocarcinoma when treatment could improve long term survival of patients.

Endoscopic investigations are invasive, and given the lack of early symptoms for GC, not generally conducted until an advanced stage where the tumour has significant invasion [2]. While routine screening by gastric photofluorography in Japan has led to an improvement in the 5YSR by over 50% [3], the costs involved cannot be justified in Western countries where the incidence is low, or in those countries with lower GDP. In addition, the sensitivity of barium X-ray reaches only 39% for early stage cancer [4], leading to this being combined with esophagogastroduodenoscopy (EGD), the combination of which is in turn limited by the number of highly experienced endoscopists and the variability of the luminous intensity and quality of endoscopic images depending on differences in endoscope diameter [5]. Unfortunately, EGD is known to be an unpleasant experience for patients invoking anxiety, pharyngeal discomfort, nausea, the gag-reflex and choking, and has been associated with adverse incidents such as cardiovascular responses [6], [7], [8].

The incidence of GC in Asia and South America is similar to that in Japan [9], and a cost effective and non-invasive test to identify individuals at risk of GC for further referral for endoscopic biopsy could contribute significantly to improving survival and reducing the health cost burden associated with this disease. Current non-invasive tests for GC include serum pepsinogen in combination with Helicobacter pylori infection testing, gastrin 17 [9], carcinoembryonic antigen (CEA), carbohydrate antigen (CA) 19–9, CA72–4 [10], and alpha-fetoprotein (AFP) [11], however, all of the above tests provide relatively low sensitivity and specificity [12], [13].

The advantages of multiple biomarkers over single biomarkers include: that protein biomarkers may be indicative of more than one disease and therefore not unique to a specific cancer e.g. Il6 is over expressed in oral, prostate, multiple myeloma and renal cell cancers [14]; single biomarkers often have inadequate predictive value e.g. about 70% for PSA [15]; a single biomarker for a given cancer may for a biochemical reason be poorly expressed in a particular patient but it is unlikely that an entire panel of protein biomarkers would fail to be expressed. It is acknowledged that at the early validation stage the use of multiple biomarkers is more expensive than a single biomarker. However, once clinical relevance is established, ELISA analysis can be integrated into routine clinical pathology platforms and multiplexed.

Few studies have looked at a suite of protein biomarkers that are differentially regulated and can show congruence of late-stage and early-stage GC biomarkers [16]. This is possibly because serum biomarkers that are able to distinguish early-stage GC are difficult to identify and validate due to the lack of serum samples from corresponding patients, the inherent difficulties of human genetic diversity and the relatively low abundance of potential serum biomarkers in comparison with the complex serum proteome [17]. Given the incidence of GC in densely populated low-income countries, a diagnostic test based on a suite of differentially regulated serum biomarkers providing good sensitivity and specificity, and that can detect early-stage GC would have international application.

Recently we reported the differential regulation of serum biomarkers in tumour-bearing versus tumour-free cohorts in a highly reproducible, preclinical validated mouse model for early stage GC [18]. We showed that the three candidate biomarker proteins afamin, clusterin and haptoglobin were individually superior to a current clinical marker CA72-4 in discriminating GC patients from healthy controls. Prior to this, and to the best of our knowledge, changes in serum levels of afamin and clusterin had not been implicated in the diagnosis of GC, thus potentially yielding novel gastric cancer biomarkers. Here we report differential regulation of 4 biomarkers, with the addition of vitamin D binding protein (VDBP) to those previously reported, in serum of an extended cohort of GC patients, including a small number (n = 11) of early gastric cancer patients. The biomarkers were validated using ELISA and MRM mass spectrometry.

Isoform specific biomarkers have been described in a number of cancers including epithelial ovarian cancer [19], and their relevance in the detection of cancer has been recently reviewed [20]. The difference in physico-chemical properties of the different isoforms has been mostly attributed to posttranslational modifications such as glycosylation. Although the identification of the modification is not absolutely necessary, it might provide important insight into the regulation of the protein of interest. The reliable quantification of protein isoforms is technically challenging and therefore limits their applicability in a clinical or diagnostic setting. However, we were able to confirm the specificity of an isoform specific biomarker using human serum by IEF-WB. Future advances in technologies are of critical importance to allow the use of this novel set of biomarkers.

The need for serum biomarkers is two-fold: firstly to reduce patient impact of screening techniques and the costs involved with such invasive measures; and secondly to enable highly specific detection of early stage GC such that treatments are highly effective thus improving %YSR's.

The well-being of patients is of utmost concern during detection and treatment of gastric and other forms of cancer at the late and early stages, and the high incidence of late stage versus early stage GC detection further highlights the need for a non-invasive, and thus low patient impact and sensitive screening technique.

Section snippets

Patient samples

Samples were collected with approval from the Ethics Committees of the Peter MacCallum Cancer Centre (Melbourne, Australia), National University Hospital (Singapore) and the University of Adelaide (Adelaide, Australia). Serum samples were obtained from 37 (female n = 10, 69 ± 10 years; male n = 17, 66 ± 11 years) preoperative GC patients with intestinal type gastric adenocarcinoma (according to Lauren classification). Eleven of these were classified as early-stage gastric cancer (AJCC 6th Edition Stage I

Candidate gastric cancer biomarker validation by ELISA

We performed ELISAs for afamin, clusterin, haptoglobin and VDBP to quantitatively assess the abundance of these proteins in patient-derived serum samples in a clinically applicable manner. Of the 18 serum samples from patients with gastric cancer, 11 were classified by AJCC criteria 6th edition as Stage I early-stage gastric cancer with minimal depth of invasion into mucosa and no metastatic lymph node involvement. All other gastric cancer patients displayed gastric cancer as Stages II–III.

Ability of serum biomarkers to identify early stage gastric cancer

Gastric cancer is most commonly detected at Stages III and IV, at which time prognosis is poor. Familial history (e.g. Lynch Syndrome and mismatch repair deficiencies) is known to play a large part in intestinal type GC with those at risk currently undergoing regular investigations. These investigations can take the form of endoscopy, or if in Japan gastric fluorography, both of which are invasive and expensive. Serum biomarkers, reporting protein profile changes as a reflection of pathological

Conclusion

In conclusion, the role of early diagnostics in the treatment of gastric cancer, as for all cancers, cannot be underestimated, thus the value in identifying an easily accessible serum biomarker. Here the ability of the circulating proteome profile to reflect the current physiological status of an individual was used to identify a suite of 4 proteins that were differentially regulated in GC versus Control cohorts. Given the silent progression of the early and treatable stages of GC to late stage

Acknowledgements

The MRM-HR analysis was undertaken at APAF the infrastructure provided by the Australian Government through the National Collaborative Research Infrastructure Strategy (NCRIS). Matthias Ernst is a senior research fellow of the NHMRC.

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    This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.

    1

    Current address: Department of Paediatrics, The University of Adelaide, Women's and Children's Hospital, SA 5006, Australia.

    2

    Current address: SA Pathology, Surgical Pathology, Bone and Joint Research Laboratory, Adelaide, SA, 5000, Australia

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