ARTICLE pubs.acs.org/jpr
Novel Glycan Biomarkers for the Detection of Lung Cancer James N. Arnold,†,‡,§ Radka Saldova,†,‡,|| Marie C. Galligan,^ Thomas B. Murphy,^ Yuka Mimura-Kimura,†,|| Jayne E. Telford,|| Andrew K. Godwin,# and Pauline M. Rudd*,†,|| †
Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, England School of Mathematical Sciences, Library Building, University College Dublin, Belfield, Dublin 4, Ireland # Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, Kansas, United States Dublin-Oxford NIBRT Glycobiology Laboratory, NIBRT, Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland
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bS Supporting Information ABSTRACT: Lung cancer has a poor prognosis and a 5-year survival rate of 15%. Therefore, early detection is vital. Diagnostic testing of serum for cancer-associated biomarkers is a noninvasive detection method. Glycosylation is the most frequent post-translational modification of proteins and it has been shown to be altered in cancer. In this paper, highthroughput HILIC technology was applied to serum samples from 100 lung cancer patients, alongside 84 age-matched controls and significant alterations in N-linked glycosylation were identified. Increases were detected in glycans containing Sialyl Lewis X, monoantennary glycans, highly sialylated glycans and decreases were observed in core-fucosylated biantennary glycans, with some being detectable as early as in Stage I. The N-linked glycan profile of haptoglobin demonstrated similar alterations to those elucidated in the total serum glycome. The most significantly altered HILIC peak in lung cancer samples includes predominantly disialylated and tri- and tetra-antennary glycans. This potential disease marker is significantly increased across all disease groups compared to controls and a strong disease effect is visible even after the effect of smoking is accounted for. The combination of all glyco-biomarkers had the highest sensitivity and specificity. This study identifies candidates for further study as potential biomarkers for the disease. KEYWORDS: N-linked glycans, lung cancer, glyco-biomarkers, sialyl Lewis X, serum
’ INTRODUCTION Lung cancer is the most common cancer worldwide and is responsible for more deaths per year than any other cancer.1 It has a poor prognosis with a 5-year survival rate of 15% and is usually diagnosed in the late stages of the disease.1 If detected in Stage IA, the 5-year survival rate increases to over 60%.2 The identification of biomarkers which are sensitive and specific to detect early stage lung cancer is vital. Biomarkers can be derived from sputum, serum and tissue.3 Serum biomarkers offer the simplest source, due to the ease of collection of blood in the clinic. Current serum protein biomarkers for lung cancer diagnosis include carcinoembryonic antigen, cytokeratin 19 fragment, tissue polypeptide antigen, progastrin-releasing peptide, neuron-specific enolase and tumor M2 pyruvate kinase.4-8 The N- and O-glycosylation patterns of serum glycoproteins have been shown to be altered in cancer and offer potential biomarkers for the disease.9,10 Two of the most well-characterized N-glycosylation changes in the serum of cancer patients are the degree of glycan branching (the number of N-acetylglucosamine (GlcNAc) residues attached to the chitobiose core) and the levels r 2011 American Chemical Society
of Sialyl Lewis X (SLex) structures.11-13 The most common Oglycosylation changes are high levels of Lewis, Tn and sialyl-Tn antigens.10,14 The SLex epitope consists of a sialic acid R2,3-linked to galactose with fucose R1,3-linked to GlcNAc and is a ligand for selectins.15 Leukocytes, which naturally express SLex epitopes, use this interaction to adhere to the endothelium and, following integrin interactions, this process results in the cells extravasating from the bloodstream. SLex levels are significantly higher on metastatic cancer cells and can be exploited by cancer cells to aid metastasis.9 As a biomarker, high levels of SLex on serum glycoproteins, and/or expression of SLex epitopes on tumor tissue are associated with a poor outcome.9,16-18 SLex is a good prognostic indicator of the stage of disease (71%), but is a weak diagnostic marker for non small cell lung cancer (24%).18 Therefore, SLex may represent a useful biomarker of tumor progression, but is a poor biomarker for disease diagnosis. More sensitive glyco-biomarkers may exist. Received: October 14, 2010 Published: January 07, 2011 1755
dx.doi.org/10.1021/pr101034t | J. Proteome Res. 2011, 10, 1755–1764
Journal of Proteome Research The serum N-glycome has previously been shown to contain 117 glycans.19 In this study, we analyzed the N-glycome of serum samples from 100 lung cancer patients and from 84 controls using hydrophilic interaction liquid chromatography (HILIC) and weak anion exchange (WAX) high performance liquid chromatography (HPLC) technology in order to identify lung cancer-related glycan alterations. This high-throughput glycan analysis technology is highly sensitive and quantitative. The glycan modifications were considered in terms of lung cancer samples vs controls and also in terms of samples from the individual stages of lung cancer vs controls, so as to determine how early these changes were detectable in the serum. In addition, the findings of this study were examined to determine whether smoking status affected the glycosylation changes.
’ MATERIALS AND METHODS Serum Samples
Serum samples from preoperative patients diagnosed with lung cancer and cancer-free healthy volunteers were obtained from Fox Chase, Cancer Center, Philadelphia, USA under IRB approved protocols. They were both from males and females. Patient sera (20 from each stage - I, II, IIIA, IIIB and IV) were examined alongside age-matched control sera from donors who did not have cancer (n = 84) (Supplementary Table S1, Supporting Information). One-Step Isolation of Haptoglobin from Serum
An affinity resin was prepared using mouse antihuman haptoglobin HG36 clone (H6395 Sigma-Aldrich). IgG was purified using a 1 mL HiTrap protein G column (Pharmacia) as previously described.20 The purified IgG (1 mg) was dialyzed into 0.1 M NaHCO3, 0.5 M NaCl, pH 8.3. An affinity resin was prepared using 0.29 g of cyanogen bromide activated Sepharose 4B (SigmaAldrich C9142) per ml of hydrated resin which was hydrated with 50 mL of 1 mM HCl for 15 min at room temperature (RT). HCl was filtered off and the 1 mL of moist resin cake was added to the dialyzed antihaptoglobin IgG (0.5 mg/mL). This mixture was stirred by slow rotation for 2 h at RT. The resin was washed with 20 mL of 0.1 M Tris, 140 mM NaCl, pH 8.0 and the volume was increased by addition of 30 mL of wash buffer and mixed by rotating for 2 h at RT to block any remaining active sites. The resin was then equilibrated in PBS-0.5 mM EDTA for storage. Haptoglobin was purified from 20 μL of serum which was diluted to 1 mL with 10 mM HEPES, 1 M NaCl, 5 mM EDTA, pH 7.4. This was subsequently incubated with 10 μL of antihaptoglobinSepharose resin and left at 4 C for 1 h at slow rotation in order to facilitate binding. The resin was pelleted by carrying out centrifugation at 1000 g and the supernatant was discarded. The resin was washed twice by resuspension in 1 mL of dilution buffer followed by centrifugation as previously described. The pellet was dissolved in 5 μL Laemmli buffer21 and 5 μL DTT (0.5M) and incubated for 5 min at 70 C before being loaded directly onto a 4-12% Bis-Tris gels (Invitrogen) for SDS-PAGE analysis. Resolved proteins were visualized using Coomassie Blue stain. Removal of N-linked Glycans from Serum and Isolated Haptoglobin
N-glycans were released from serum using the high-throughput method described by Royle et al.,.19 Briefly, serum samples were reduced and alkylated in 96-well plates, then they were immobilized in SDS-gel blocks and were washed. The N-linked glycans were released using peptide N-glycanase F (1000 U/mL; EC 3.5.1.52) as described previously.22,23 N-glycans were
ARTICLE
released from isolated haptoglobin using the in-gel block method from SDS-PAGE bands.24 2-Aminobenzamide (2-AB) Labeling of Glycans
Released glycans were labeled by reductive amination with the fluorophore 2-AB,22 using a Ludger Tag 2-AB glycan labeling kit as described by the manufacturer (Ludger Ltd., Oxford, U.K.). Excess 2-AB reagent was removed by ascending chromatography on Whatman 3MM paper (Clifton, NJ) in acetonitrile. HILIC
HILIC was performed using a TSK-Gel Amide-80 4.6 250 mm column (Anachem, Luton, Bedfordshire, UK) on a 2695 Alliance separation module (Waters, Milford, MA) equipped with a Waters temperature control module and a Waters 2475 fluorescence detector. Solvent A was 50 mM formic acid which was adjusted to pH 4.4 with ammonia solution. Solvent B was acetonitrile. The column temperature was set to 30 C. Gradient conditions were as follows: 180 min method (isolated haptoglobin)-a linear gradient of 20-58% A, over 152 min at a flow rate of 0.4 mL/min; 60 min method (serum samples) - a linear gradient of 35 to 47% solvent A over 48 min at a flow rate of 0.8 mL/min, followed by 1 min at 47 to 100% A and 4 min at 100% A, returning to 35% A over 1 min and then finishing with 35% A for 6 min.19 Samples were injected in 80% acetonitrile.24 Fluorescence was measured at 420 nm with excitation at 330 nm. The system was calibrated with a dextran ladder, as described previously.24 WAX-HPLC
WAX-HPLC was performed using a Vydac 301VHP575 7.5 50mm column (Anachem) on a 2695 Alliance separations module with a 474 fluorescence detector (Waters). Solvent A was 0.5 M formic acid adjusted to pH 9.0 with ammonia solution, and solvent B was 10% (v/v) methanol in water. Gradient conditions were as follows: a linear gradient of 0 to 5% A over 12 min at a flow rate of 1 mL/min, followed by 5-21% A over 13 min and then 2-50% A over 25 min, 80-100% A over 5 min followed by 5 min at 100% A. Samples were injected in water. A fetuin N-glycan standard was used for calibration24 All HPLC units were equipped with Waters temperature control modules and Waters 2475 fluorescence detectors set with excitation and emission wavelengths of 330 and 420 nm, respectively.19 Exoglycosidase Digestions
All enzymes were purchased from Prozyme (San Leandro, CA). The 2-AB-labeled glycans were digested in 10 μL of 50 mM sodium acetate buffer, pH 5.5 for 18 h at 37 C, using arrays of the following enzymes at the indicated concentrations: ABS, Arthrobacter ureafaciens sialidase (EC 3.2.1.18), 1 U/mL; BTG - Bovine testes β-galactosidase (EC 3.2.1.23), 1 U/mL; BKF, Bovine kidney R-fucosidase (EC 3.2.1.51), 1 U/mL; AMF, Almond meal R-fucosidase (EC 3.2.1.111), 3 mU/mL. After incubation, enzymes were removed by filtration through protein-binding EZ filters (Millipore Corporation).24 N-glycans were then analyzed by HILIC or WAX-HPLC. CRP ELISA
CRP was measured in all samples using High sensitivity CRP ELISA kit (Foster City, CA). Statistics
50-50 ANCOVA/MANCOVA25 were carried out to determine the factors influencing the peaks of interest, using the R software package26 and the add-on package fmanova.27 Tukey’s HSD “Honestly significant difference” test was performed to calculate p-values, which were adjusted for multiple testing to make pairwise comparisons between levels of a significant grouping factor. Multivariate 1756
dx.doi.org/10.1021/pr101034t |J. Proteome Res. 2011, 10, 1755–1764
1757 0.27 ( 0.14 7.26 ( 2.62 0.47 ( 0.26 1.74 ( 0.44
0.19 ( 0.08 0.31 ( 0.27 1.47 ( 0.27
Log SLexe
N/A
0.81
1.00
stage I
1.09 ( 0.63
0.79 ( 0.55
0.79 ( 0.61
0.09
0.56 ( 0.75
0.37 ( 0.6
Log CRPe
0.77 ( 0.62
0.59
0.76 ( 0.38
N/A
62.94 ( 2.30 61.60 ( 2.42 62.06 ( 2.75 61.48 ( 2.19 61.21 ( 2.39 62.43 ( 2.49 61.76 ( 2.44 15.68 ( 2.39 16.68 ( 2.59 17.79 ( 2.81 17.92 ( 3.07 18.61 ( 2.45 17.26 ( 2.24 17.65 ( 2.67
0.12
0.11
0.03
0.09
0.03
1.88 ( 0.53
0.53 ( 0.25
7.44 ( 2.33
0.27 ( 0.16
6.26 ( 2.03
control-
0.08
0.02
N/A
