Serum N-Glycome Characterization in Patients with Resectable

Article pubs.acs.org/jpr

Serum N‑Glycome Characterization in Patients with Resectable Periampullary Adenocarcinoma Julian Hamfjord,† Radka Saldova,⊥ Henning Stöckmann,⊥ Vandana Sandhu,†,□ Inger Marie Bowitz Lothe,†,‡ Trond Buanes,§,¶ Ole Christian Lingjærde,#,∥ Knut Jørgen Labori,§ Pauline M. Rudd,⊥ and Elin H. Kure*,†,□ †

Department of Cancer Genetics, Institute for Cancer Research, ‡Department of Pathology, and §Department of Hepato-Pancreato-Biliary Surgery, Oslo University Hospital, 0424 Oslo, Norway ⊥ NIBRT GlycoScience Group, The National Institute for Bioprocessing Research and Training, Dublin, Ireland ¶ Institute of Clinical Medicine, Faculty of Medicine, #Department of Computer Science, and ∥K.G. Jebsen Centre for Breast Cancer Research, Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, 0316 Oslo, Norway □ Department of Environmental and Health Studies, Faculty of Arts and Sciences, Telemark University College, 3800 Bo in Telemark, Norway S Supporting Information *

ABSTRACT: Serum N-glycans are promising biomarkers for systemic disease states. Better understanding of the serum N-glycome of patients with resectable periampullary adenocarcinoma may identify novel prognostic markers for this disease. Serum N-glycans in 70 patients with resectable periampullary adenocarcinoma, 15 patients with benign periampullary tumor, and 129 healthy individuals were quantified using ultra performance liquid chromatography. High-sensitivity C-reactive protein (hsCRP) was analyzed for all samples using an immunoturbidimetric method. The N-glycome was compared to clinical and histopathological data, and to the acute phase response as measured by hsCRP. Whole-genome tumor tissue mRNA expression data were used for correlation and enrichment analysis to investigate underlying biological processes giving rise to changes in the serum N-glycome. Significant changes were found in the serum N-glycome of patients with periampullary adenocarcinoma (n = 70) compared to healthy individuals (n = 129). No significant differences were found between patients with benign (n = 15) and malignant periampullary tumors (n = 70). Many alterations in the N-glycome correlated with systemic acute phase response as measured by hsCRP. Enrichment analysis indicated that immunologic pathways of the cancer microenvironment correlate with specific features of the serum N-glycome. Certain glycans were associated with poor overall and disease free survival in patients with pancreatobiliary type of periampullary adenocarcinoma. Our study supports the hypothesis that certain factors secreted by the tumor affect liver and plasma cells to orchestrate the changes in the serum N-glycome observed. The serum N-glycome could potentially reflect modified phenotypes of the host and/or tumor microenvironment. The prognostic impact of the serum Nglycome should be evaluated in larger, prospective studies. KEYWORDS: periampullary adenocarcinoma, pancreatic cancer, pancreatitis, N-glycan, inflammation, acute phase response

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INTRODUCTION Periampullary adenocarcinoma may originate from the pancreas, the distal common bile duct, the ampulla of Vater, or the duodenum. The majority of patients present at a late stage and have locally advanced or metastatic disease at the time of diagnosis. Tumors originating from the pancreas have the worst prognosis. Some patients are candidates for curative surgery, and resectable tumors are all given the same surgical treatment. The origin of the tumor has until recently been a major stratification determinant for the type of adjuvant chemotherapy given to these patients. There is, however, recent evidence that morphologic differentiation (intestinal or pancreatobiliary) is a better predictor for survival1,2 and may © 2015 American Chemical Society

open for a change in current treatment protocols for periampullary adenocarcinoma.3,4 Owing to the poor prognosis of these diseases, considerable effort has been made to identify prognostic biomarkers in serum.5 Still, only the serum glycoprotein CA19−9 has so far made it into clinical use. In the search for new biomarkers, studies have been performed for the detection of markers in both tissues and serum. Glycosylation is the most common post-translational modification in eukaryotes. The collection of sugar chains Received: May 14, 2015 Published: October 30, 2015 5144

DOI: 10.1021/acs.jproteome.5b00395 J. Proteome Res. 2015, 14, 5144−5156

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Journal of Proteome Research Table 1. Clinical and Histopathology Data of Patients with Periampullary Adenocarcinomaa intestinal adenocarcinoma (n = 13) median age (years) gender origin

differentiation

tumor

node

metastasis resection vessel infiltration perineural infiltration recurrenceb dead DFS (days)f OS (days)f

count

%

7 6 7 0 5 1 1 0 12 1 7 2 3 8 5 0 13 12 1 8 5 9 4 7 5 9 4

53.8% 46.2% 53.8% 0.0% 38.5% 7.7% 7.7% 0.0% 92.3% 7.7% 53.8% 15.4% 23.1% 61.5% 38.5% 0.0% 100.0% 92.3% 7.7% 61.5% 38.5% 69.2% 30.8% 58.3% 41.7% 69.2% 30.8%

62

pancreatobiliary adenocarcinoma (n = 57) median

count

%

29 28 6 12 0 39 1 25 31 4 5 47 1 19 37 1 57 32 25 22 35 3 54 5 52 8 49

50.9% 49.1% 10.5% 21.1% 0.0% 68.4% 1.8% 43.9% 54.4% 7.0% 8.8% 82.5% 1.8% 33.3% 64.9% 1.8% 100.0% 56.1% 43.9% 38.6% 61.4% 5.3% 94.7% 8.8% 91.2% 14.0% 86.0%

67

F M Ampulla of Vater† Distal bile duct† Duodenum† Pancreas† High Medium† Low† T1 T2† T3† T4† N0 N1 N2 M0 R0† R1† No† Yes† No† Yes† No† Yes† No† Yes† 1362 1633

329 533

a

Comparison of means (t test) and proportions (z-tests) where significantly different values at p < 0.05 (corrected for multiple testing using Bonferroni method) are denoted with subscripts f and †, respectively. bOne patient died of stroke, and recurrence data is not recorded.

phosphates, and acetates. This makes glycans technically more difficult to study compared to other macromolecules like DNA, RNA, and proteins. Yet, high-throughput analysis of the serum N-glycome has improved over the past few years, making the methods attractive for new biomarker discovery. In this study, we have utilized a method that can give reproducible global serum N-glycome representations within a reasonable time frame, making the method ideal for studying larger series of human samples. The serum N-glycome of 70 patients with periampullary adenocarcinoma and 15 patients with nonmalignant periampullary tumor has been compared to that of the serum profiles of 129 healthy individuals. The Nglycome has been compared to clinical and histopathological data. Additionally, whole-genome mRNA expression of periampullary adenocarcinoma has been correlated with Nglycan levels to investigate the underlying biological processes giving rise to changes in the serum N-glycome in this patient group.

(glycans) synthesized by a cell under specific conditions is called its glycome, and aberrant glycomes have been identified in both tissue and serum of patients with cancers6−11 and systemic inflammation.9,12 The best characterized N-glycome alterations in malignant cells are the increased branching of sugar moieties and higher levels of the Sialyl-LewisX (SLeX) antigens present. This may induce changes in protein structure, stability, and affinity due to alterations in mass, shape, charge, or other physical properties. The biological implications of such alterations are only partially known. For instance, the epidermal growth factor receptor (EGFR) will be activated for a longer time period given an increased N-glycan branching.13 This leads to the prolonged maintenance of the receptors on the cell membrane, increased signaling activation, cell growth, and division. Another notable example is that pancreatic adenocarcinoma cell lines overexpressing SLeX have a greater metastatic potential compared to cells where the antigen has been blocked,14,15 probably due to alterations in cell−cell and cell−extracellular matrix communication. The serum Nglycome has also been partly described for several cancers,9 but less is known about the functional role of these alterations. Glycans are structurally complex in nature since one protein may have many glycosylation sites, there are multiple monosaccharide combinations, there is anomeric linkage position, there may be extensive branching, and glycans may include noncarbohydrate substituents such as sulfates,

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MATERIALS AND METHODS

Patients and Specimens

A total of 85 patients with periampullary tumors admitted to Oslo University Hospital for pancreatoduodenectomy with curative intent (2008−2011) were included in the study. Serum was collected prior to surgery, and available fresh frozen tumor 5145


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Nanodrop ND-1000 spectrophotometer and the quality assessed on a Bioanalyzer 2100 (Agilent).4

tissue was sampled from surgical specimens. Patients with evidence of distant metastases before or during surgery were not included in the project. The histopathological examination followed a standardized protocol for pancreatoduodenectomy specimens. The diagnosis was independently verified by two experienced pathologists according to the WHO Classification of Tumors of the Digestive System 2010. The adenocarcinomas were classified in accordance with the pTNM Classification of Malignant Tumours, seventh edition. Adenocarcinomas were further subclassified into pancreatobiliary and intestinal type.16 All adenocarcinomas from the periampullary region (57 pancreatobiliary and 13 intestinal type) were included in this project. Tumors that were of mixed pancreatobiliary and intestinal types were classified according to the dominant subtype. Additionally, 15 tumors were of a benign histopathology (2 serous cystadenomas, 7 intraductal papillary mucinous neoplasms, and 6 pancreatitis). A summary of clinical and histopathology data for patients with adenocarcinomas is presented in Table 1. The healthy control group consisted of men and women from a Danish and Norwegian cohort, respectively. The women were part of a Norwegian study aimed at exploring breast cancer risk factors. Eligible women were those referred to breast diagnostic centers due to findings on the screening mammogram or due to clinical findings. A total of 169 women were included, 107 of these with no breast malignancy, hence controls.17 A total of 61 women were randomly selected for the control cohort. The men were part of a Danish study on the effect of farmed trout on cardiovascular risk markers in healthy men.18 A total of 68 men were randomly selected for the control cohort. Demographic data for all patients and healthy controls are presented in Table 2.

mRNA Microarray

One-hundred nanograms total RNA was converted to cDNA, amplified, and labeled with Cy-3 using LowInput QuickAmp Labeling Kit (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s instructions. After the samples were cleaned on RNeasy Mini Columns (Qiagen), the efficiency of the labeling reaction labeled cRNA was measured using Nanodrop ND-1000. Six-hundred nanograms of Cy-3 labeled cRNA was hybridized to SurePrint G3 Human GE 8 × 60K microarrays for 17 h at 65 °C using Hi-RPM Gene Expression Hybridization Kit (Agilent Technologies, Santa Clara, CA, USA). The arrays were washed according to the manufacturer’s instructions, using Gene Expression Wash Buffer Kit (Agilent Technologies, Santa Clara, CA, USA), and scanned on an Agilent DNA Microarray Scanner. Hybridization signals were extracted using Feature Extraction 10.7.3.1 (Agilent Technologies, Santa Clara, CA, USA). The mRNA microarray data were background corrected and quantile normalized, and are published in a public repository.4 The data are accessible through GEO accession number GSE60980. A total of 56 malignant cases passed quality control and were used in the subsequent analysis. C-Reactive Protein Analyses

High-sensitivity C-reactive protein (hsCRP) was analyzed using the QuikRead go system (Orion Diagnostica, Espoo, Finland), which uses an immunoturbidimetric method. In brief, 12 μL of serum sample was added to the buffer solution in the cuvette and analyzed according to the instructions of the manufacturer. In the case of a hsCRP value >75 mg/L, samples were diluted in 0.9% saline and reanalyzed. A positive control was analyzed whenever a new kit was introduced.

Table 2. Demographic Data for Patients with Malignant Tumors, Benign Tumors, and Healthy Controlsa

age (median years) gender F M BMI (kg/m2) any current disease any current medication current smoking a

malignant (n = 70)

benign (n = 15)

controls (n = 129)

66 51% 49% 23.7 63% 61%

64 27% 73% 25.2 60% 47%

52 47% 53% 24.5 N/A N/A

30%

54%

N/A

N-Glycan Analyses

N-glycans were released from 5 μL of serum samples using the high-throughput automated method previously described by Stöckmann et al.19 modified for serum samples.20 Briefly, the samples were denaturated with dithiothreitol, alkylated with iodoacetamide, and N-glycans were released from the protein backbone enzymatically via PNGase F (Prozyme Glyco NGlycanase, code GKE-5006D, 10 μL per well, 0.5 mU in 1 M ammonium bicarbonate, pH 8.0). Next, glycans were immobilized on solid supports, and excess reagents were removed by vacuum or centrifuge filtration. Glycans were released from the solid supports and labeled with the fluorophore 2-aminobenzamide (2-AB). Next, glycans were cleaned up using 96-well chemically inert filter plate (Millipore Solvinert, hydrophobic polytetrafluoroethylene membrane, 0.45 μm pore size) using HyperSep Diol SPE cartridges (Thermo Scientific).19 All samples were analyzed in duplicates.

N/A, not available.

Blood samples were collected according to local procedures and may differ slightly between the cohorts. In general, effort has been made to process most samples within one working day. Serum has been frozen at −80 °C. The research program is approved by the Regional Ethical Committee for registration of clinical data and sampling of blood and tumor tissue for research purposes (REK 265− 08412c). The included patients and healthy individuals have all signed informed consent.

Ultra Performance Liquid Chromatography (UPLC)

UPLC was performed using a BEH Glycan 1.7 μm particles in 2.1 × 150 mm column (Waters, Milford, MA) on an Acquity UPLC (Waters, Milford, MA) equipped with a Waters temperature control module and a Waters Aquity fluorescence detector. Solvent A was 50 mM formic acid adjusted to pH 4.4 with ammonia solution. Solvent B was acetonitrile. The column temperature was set to 40 °C. The 30 min method was used with a linear gradient of 30−47% with buffer A at 0.56 mL/min flow rate for 23 min followed by 47−70% A and finally reverting back to 30% A to complete the run method. Samples

Isolation of RNA

Total RNA was isolated from 10 × 20 μm sections of fresh frozen tumor tissue using mirVana miRNA Isolation Kit (Ambion/Life Technologies) according to the manufacturer’s instructions. RNA concentration was measured using a 5146


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Figure 1. List of N-glycan structures represented in each peak and estimated features. Structure abbreviations: All N-glycans have two core GlcNAcs; F at the start of the abbreviation indicates a core-fucose α1,6-linked to the inner GlcNAc; Mx, number (x) of mannose on core GlcNAcs; D1 indicates that the α1−2 mannose is on the Manα1−6Manα1−6 arm, D2 on the Manα1−3Manα1−6 arm, D3 on the Manα1−3 arm of M6, and on the Manα1−2Manα1−3 arm of M7 and M8; Ax, number of antenna (GlcNAc) on trimannosyl core; A2, biantennary with both GlcNAcs as β1,2linked; A3, triantennary with a GlcNAc linked β1,2 to both mannose and the third GlcNAc linked β1,4 to the α1,3 linked mannose; A4, GlcNAcs linked as A3 with additional GlcNAc β1,6 linked to α1,6 mannose; B, bisecting GlcNAc linked β1,4 to β1,3 mannose; Gx, number (x) of β1,4 linked galactose on antenna; F(x), number (x) of fucose linked α1,3 to antenna GlcNAc; Sx, number (x) of sialic acids linked to galactose; Lac(x), number (x) of lactosamine (Galβ1−4GlcNAc) extensions. ∗, Sialic acids isomers (same composition but different sialic acid linkage arrangements resulting in different glucose units (GU) from the original structures). Peaks calculated into specific features are highlighted in colors. Certain peaks only 5147


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Journal of Proteome Research Figure 1. continued

contribute with an approximate fraction to a certain feature, labeled in figure as (a) 33%, (b) 50%, or (c) 66%. Sialylation: S0 (neutral, GP1−15); S1 (monosialylated, GP16−20); S2 (disialylated, GP21−26); S3 (trisialylated, GP27−32); S4 (tetrasialylated, GP33−38). Galactosylation: G0 (agalactosylated, GP1−2+GP4−6); G1 (monogalactosylated, GP3+GP7−11+(GP13/2)+GP16+(GP18/2)); G2 (digalactosylated, (GP13/ 2)+GP14−15+GP17+(GP18/2)+GP19−24); G3 (trigalactosylated, GP25−30); G4 (tetragalactosylated, GP31−38). Branching: A1 (monoantennary, GP1−3+(GP7*2/3)+(GP13/2)+(GP18/2)); A2 (biantennary, GP4−5+(GP6/2)+(GP7/3)+GP8−11+(GP13/2)+GP14−17+GP18/ 2+GP19−24); A3 (triantennary, GP25−30); A4 (tetraantennary, GP31−38). High mannose: (GP6/2)+GP12. Fucosylation: Core-fucose, GP2+GP5+(GP6/2)+(GP7*2/3)+GP9−11+GP15+(GP16/2)+GP19−20+GP23−24+GP29+(GP36/2) and outer arm fucose, GP30+GP32+(GP33/3)+GP37+(GP38/3).

where both data sets were available (n = 56). In brief, all array spots containing hybridization probes (n = 42 543) were reduced to only known gene loci (n = 29 665). Since one gene may be represented in more than one array spot, an average expression of identical genes was calculated. This reduced the number of unique gene expressions (n = 20 740). Then, correlations for all log1p transformed glycan peaks and quantile normalized genes were calculated. Genes with significant correlations and anticorrelations for each glycan peak (p < 0.05) were identified. A downstream gene enrichment analysis was performed using an R integration of the Database for Annotation, Visualization, and Integrated Discovery (DAVID version 6.7) in the package RDAVIDWebService. Enriched KEGG pathways with p < 0.00042 (Bonferroni-adjusted p < 0.05) were selected and visualized using the package BACA in R.

were injected in 70% acetonitrile. Fluorescence was measured at 420 nm with excitation at 330 nm. The system was calibrated using an external standard of hydrolyzed and 2AB-labeled glucose oligomers to create a dextran ladder, as described previously.21 Survival Analysis

Overall survival (OS) was calculated from date of surgery to date of death. OS data were obtained from the National Population Registry in Norway. Disease free survival (DFS) was calculated from date of surgery to date of recurrence of disease. Recurrence was defined as radiological evidence of intraabdominal soft tissue around the surgical site or of distant metastasis. Final date of data collection was December 22, 2014. OS and DFS were only evaluated in patients with malignant disease.

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Statistics and Bioinformatics

RESULTS The global serum N-glycome was separated into 38 peaks and assigned according to Saldova et al.;17 it is summarized in Figure 1 and Table S2. An example of a representative serum sample HILIC-UPLC chromatogram from a healthy control and a patient with periampullary adenocarcinoma is shown in Figure S1. The glycan features of total core fucose, outer arm fucose, and highly mannosylated glycans in addition to degree of sialylation (S0−S4), galactosylation (G0−G4), and branching (A1−A4) were calculated based on the composition of glycan peaks 1−38 (Figure 1).

Statistical analysis was performed using the SPSS statistical software for Windows (version 21; SPSS Inc., Chicago, IL) and R (version 3.0.3). Glycan peak abundance data represent the relative percentage areas derived from the HILIC-UPLC profiles using the Empower Chromatography Data Software (Waters, MA, USA). The data are compositional, and the sum of the areas is 100%. The data are further log10 transformed to map onto a more Gaussian distribution. One-way ANOVA (analysis of variance) was used to test for associations between glycan peak areas and various clinical, biochemical, and histopathological parameters. Results are reported as differences in log transformed relative abundances and only as significant if the adjusted p < 0.05. Correction for multiple testing is performed using a simple Bonferroni correction with a desired significant threshold of p < 0.05. For glycan peaks, this translates to an unadjusted p-value of 0.05/38 = 0.001 and for glycan features to an unadjusted p-value of 0.05/17 = 0.003. The log rank (Mantel-Cox) test of equality of survival distributions was used to compare the survival of patients with glycan peak area above and below the median area. The median was chosen as an unbiased threshold for stratification. The Kaplan−Meier estimators of survival were plotted, and p-values from the log rank tests were reported. The prediction analysis for microarrays (PAM) was utilized to investigate to what degree serum glycans can separate individuals with malignant and benign periampullary tumors as well as malignant and healthy individuals. PAM uses the nearest shrunken centroid method22 and is provided in the package pamr using R. Hierarchical clustering of all serum samples was performed using complete linkage and Spearman’s rank correlation for log10 transformed glycan peaks. A heatmap was plotted using the package gplots in R. Pearson’s correlations were calculated between gene expression and serum glycan levels within a subset of patients

Demographic Differences

There are considerable differences in serum N-glycans of males compared to females among healthy individuals, with significant differences in GPs 1−6, 9, 10, 13, 17−18, and 23 (Bonferroniadjusted p < 0.05). Less pronounced variations are seen with age, as only GP 19 shows significantly different levels in old versus young individuals (Bonferroni-adjusted p < 0.05). The variability of the N-glycome with regard to age and gender has previously been well described and generally accounts for less than 10% of the variation observed.23 In the current study, GP 19 containing bigalactosylated, biantennary, monosialylated glycans with core-fucose (FA2G2S1) decreases significantly with age in healthy controls, which is consistent with previous reports.23,24 Glycan peaks GPs 1−6 and GPs 9−10 are higher in men compared to women in healthy controls, whereas GPs 13, 17, and 18 are higher in women than men. The gender differences are consistent with previous reports.24 Major Changes Observed in Serum N-Glycans of Patients with Periampullary Adenocarcinoma

All peaks except for GPs 9, 10, 14, 17, 22, 26, 27, 29, and 38 were found to be significantly different (Bonferroni-adjusted p < 0.05) when comparing the malignant serum levels with that of controls (Figure S2). GP 4 and GP 12 are the glycan peaks 5148


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Figure 2. Unsupervised hierarchical clustering of all patients in the study versus serum N-glycan peaks (GP) 1−38, where rows represent GPs and columns represent samples. The figure clearly shows two clusters of patients with malignant (red) and benign (yellow) periampullary tumors versus healthy individuals (green). Row Z-scores are indicated in mainframe, where high levels of a given glycan peak are red, and low levels of a peak are blue. Complete linkage together with Spearman’s rank correlation was used.

Figure 3. Box-plot of sialylation, galactosylation, and branching of N-glycans in sera of patients with malignant and benign tumors, and healthy individuals (controls). The levels of S0, G0, A1, S4, G4, and A4 are all higher in serum from patients with periampullary adenocarcinoma compared to healthy individuals (Bonferroni-adjusted p < 0.05). There are no significant differences between sera of patients with malignant and benign periampullary lesions.

(A3) are significantly different in sera from patients with periampullary adenocarcinoma compared to that of healthy individuals (Bonferroni-adjusted p < 0.05). An overall increase in complex N-glycans is evident by the increase in highly sialylated, galactosylated, and branched structures. Common for these structures is the complexity and increasing size, and these structures are typically represented at the end of the chromatogram (Figure S1). Furthermore, there is an overall increase in simple N-glycans as evident by the increase in monoantennary, asialylated, and agalactosylated structures. These structures are typically represented in the beginning of

with the most profound differences, with higher levels in serum from cases. The PAM analysis, however, demonstrates that at least 13 glycan peaks are needed to lower the misclassification error to an acceptable level of 1% (Figure S2). Hierarchical clustering of all serum samples clearly demonstrates one major cluster for cases compared to that of the healthy controls (Figure 2). Both Simple and Complex N-Glycans Are Increased in Periampullary Adenocarcinoma Serum

When evaluating glycan features, all except the monogalactosylated (G1), trigalactosylated (G3), and triantennary structures 5149


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Figure 4. Box-plot of high mannose, core fucose, and outer arm fucose of N-glycans in sera of patients with malignant and benign tumors, and healthy individuals (controls). The level of high mannose is higher in serum from patients with periampullary adenocarcinoma compared to healthy individuals (Bonferroni-adjusted p < 0.05). The level of core fucose is lower and outer arm fucose is higher in patients with periampullary adenocarcinoma compared to healthy individuals (Bonferroni-adjusted p < 0.05). There are no significant differences between sera of patients with malignant and benign periampullary lesions.

37, whereas strongest negative correlation is found for GPs 19 and 21 (absolute Pearson’s r > 0.5 and Bonferroni-adjusted p < 0.05). Glycans showing strong positive correlation with hsCRP are complex, tetraantennary, tetrasialylated, and tetragalactosylated structures typically with outer arm fucose.

the chromatogram. There are no significant differences in glycan features between individuals with malignant and benign periampullary lesions (Figure 3). Fucosylation and Mannosylation Are Altered in Periampullary Adenocarcinoma Serum

Serum N-Glycome and Association with Survival of Periampullary Adenocarcinoma Patients

Fucosylation pattern is significantly altered in periampullary adenocarcinoma serum, with outer arm fucose increased and core fucose decreased. Highly mannosylated structures are increased in malignant and benign sera compared to healthy individuals. There are no significant differences in glycan features between individuals with malignant and benign periampullary lesions (Figure 4).

Median DFS for pancreatobiliary type of periampullary adenocarcinoma is significantly different for high and low levels of GP 1 (297 and 394 days, respectively; p = 0.011). Median OS is also significantly different (481 and 773 days, respectively; p = 0.009) (Figure 5). GP 1 contains monoantennary, agalactosylated, and asialylated structures. The median of GP 1 in patients with pancreatobiliary type of periampullary adenocarcinoma is 0.20. For comparison, the mean of GP 1 in healthy individuals is 0.13 (SD 0.045), in patients with benign lesions 0.70 (SD 0.85), and in patients with malignant periampullary adenocarcinoma 0.89 (SD 1.3). The same trend in survival holds when considering the feature of monoantennary structures (A1), where high levels are associated with poorer DFS (p = 0.042) and OS (p = 0.015). High levels of monoantennary structures (A1) are also associated with poorer OS when considering the periampullary adenocarcinoma of both intestinal and pancreatobiliary type (p = 0.036). An additional observation is that high levels of GP 17 are significantly associated with improved OS (p = 0.009) but not with DFS in patients with pancreatobiliary type of periampullary adenocarcinoma (data not shown). These findings do not fully reach statistical significance with stringent correction for multiple testing. No association is seen between preoperative hsCRP levels and DFS (p = 0.15) or OS (p = 0.38) when stratifying hsCRP levels for pancreatobiliary type of periampullary adenocarcinoma above and below 10 mg/ L (data not shown). Both OS and DFS are independent of gender when evaluating patients with pancreatobiliary type of periampullary adenocarcinoma (data not shown). Stratified survival analysis for the intestinal subtype is not performed due to the low sample size.

There Are No Detectable Differences in Serum N-Glycan Profiles of Benign and Malignant Periampullary Tumors

There are no statistical differences in levels of serum glycans between benign and malignant periampullary tumors. Unsupervised hierarchical clustering of samples shows that glycan expression patterns of the two groups are similar (Figure 2). Furthermore, the supervised PAM analysis shows that there is no glycan classifier that can discriminate the two groups. There Are Significant Differences in Levels of Glycan Peaks GP 33 and GP 35 and Tumor Localization

The lowest levels of GP 33 and GP 35 are found in periampullary adenocarcinoma originating from the ampulla, whereas the highest levels are found in adenocarcinoma originating from the duodenum (Bonferroni-adjusted p < 0.05). Levels of these glycans are similar in adenocarcinoma originating from the pancreas and the bile duct. There Are No Significant Associations between Histological Subtype and Serum N-Glycan Levels of Periampullary Adenocarcinoma Patients

No other associations are found between glycan peaks or features and histological subtype, tumor size, lymph node status, microscopic resection margin status, vessel infiltration, perineural infiltration, or KRAS mutation status in patients with malignant disease. Serum N-Glycome Is Highly Correlated with Systemic Acute Phase Response As Measured by hsCRP

Immune Regulatory Pathways Are Enriched in Periampullary Adenocarcinoma and Correlate with Serum N-Glycan Features

Most of the serum N-glycome is significantly correlated (Bonferroni-adjusted p < 0.05) with high-sensitivity C-reactive protein (hsCRP). This includes GPs 4−6, 12, 15, 16, 19−22, 24, 25, 28, 30−34, and 36−38 as well as features S1, S3, S4, G0, G2, G4, A2, A4, high mannose, core, and outer arm fucose. Strongest positive correlation is found for GPs 30−34 and 36−

Whole genome expression data (n = 20 740 unique gene annotations) from 56 adenocarcinoma tissue samples were correlated with serum N-glycan peaks (n = 38), and an enrichment analysis was performed. An abundance of enriched 5150


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pathways are related to local immune responses in the tumor and correlate with specific glycan peak levels, especially GP 33, GP 34, and GP 36 (Figure 6). More specifically, there is a significant enrichment of genes related to T- and B-cell receptor signaling, natural killer cell mediated cytotoxicity, antigen processing and presentation, chemokine signaling pathways, and cytokine-cytokine receptor interaction (Table S1). It is noted that complex glycans in particular are positively correlated with genes involved in local immune response. There are also enriched pathways related to cell adhesion, extracellular matrix receptor interaction, and focal adhesion with gene sets being both positively and negatively correlated to different serum N-glycan structures (Table S1).

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DISCUSSION The current study demonstrates that the serum N-glycome of patients with resectable periampullary adenocarcinoma is considerably altered compared to healthy individuals. Particularly pronounced is the increase in sialylated, galactosylated, and branched glycans, and the reduction of some less decorated structures. This result, and especially the increased glycan branching and sialylation, is consistent with most studies irrespective of cancer type.7,10,17,25−27 Furthermore, we observed decreased core fucose and increased outer arm fucose in the periampullary adenocarcinoma serum N-glycome. Similar trends with decreased core fucose have been observed in serum of gastric,28,29 colorectal,30 and lung cancer patients.8 Few studies exist on the prognostic role of the serum Nglycome, and to the best of our knowledge, this is the first study to present data from a cohort of resectable periampullary adenocarcinoma prior to surgery. A recent study on pancreatic cancer, including patients with both localized and metastatic disease, investigated the prognostic role of the serum Nglycome.26 That study identified certain structures associated with poor survival, notably a triantennary complex structure. Similar findings have been reported in renal cell carcinoma.27 We found that monoantennary structures are associated with poor survival of patients with nonmetastatic pancreatobiliary adenocarcinoma, although the results are not significant when applying stringent correction for multiple testing. More specifically, high levels of a monoantennary, agalactosylated,

Figure 5. Disease-free survival (DFS) and overall survival (OS) given high and low levels of glycan peak 1 (GP 1) for the study population of pancreatobiliary type of periampullary adenocarcinoma (n = 57), in panels A and B, respectively. Model structure for GP 1 is indicated in panel A.

Figure 6. KEGG pathway enrichment analysis of correlation between tissue mRNA expression and serum N-glycan peaks. Involved enriched pathways are listed in each row, where significant positive correlations are illustrated with a red bubble, and negative correlations with a green bubble. The size of the bubble indicates the number of genes enriched in a given correlation. There is an enrichment of immunological pathways such as Tand B-cell receptor signaling, natural killer cell mediated cytotoxicity, antigen processing, and cytokine signaling. 5151


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Figure 7. Hypothesized relationship between key players in serum N-glycome expression of periampullary adenocarcinoma. The liver and plasma cells are the main contributors of serum proteins. Many of these proteins, including acute phase proteins and immunoglobulins (Ig) are known to be N-glycosylated. Similar overall alterations in the serum N-glycome are observed both in patients with malignant processes and inflammatory diseases, for instance in pancreatic cancer and pancreatitis. Simpler N-glycan structures in the far left of the chromatogram (S0, G0−1, A1) have been shown in previous studies to be associated with immunoglobulins, whereas more complex N-glycan structures in the far right of the chromatogram (S2−4, G3−4, A4) have been shown to be associated with acute phase proteins. Lower sialylation and galactosylation on IgG have been shown to modify the immunophenotype, have increased complement-dependent cytotoxicity, and be associated with cancer progression. On the other hand, it has also been demonstrated that increased branching, sialylation, and galactosylation on certain acute phase proteins may lead to a longer half-life of these proteins. The proteins will then stay in the circulation for a longer time period and may exhibit anti-inflammatory properties. We hypothesize that altered immune signaling in the tumor and its microenvironment will produce certain factors (e.g., cytokines) that may act in an endocrine fashion and induce effects in the liver and plasma cells. Our correlation analysis points to certain potential players of the microenvironment of the tumor (Figure 6 and Table S1). The resulting modified host response may, as exemplified above, give tumor cells both the hypothetical advantage and disadvantage of growth, invasiveness, and metastasis.

phase response, as measured by serum hsCRP. This suggests that the changes of the serum N-glycome are partially derived from the liver and reflect a systemic inflammatory response. Glycoproteomic studies on serum of cancer patients do in fact show that acute-phase proteins such as α 1 -acid glycoprotein, α1-antichymotrypsin, haptoglobin, and transferrin are important contributors to the altered glycome observed.7,10,11,34 Increases in high mannose serum N-glycans are found in patients with pancreatic cancer as well as in patients with benign lesions of the pancreas, as demonstrated in our study. High mannose structures are likely associated with complement factor C3, as this is the only acute phase protein that carries this feature.35 We can also report a substantial and significant increase in the core fucosylated agalactosylated biantennary glycan structure (FA2) in individuals with periampullary tumors. This structure has previously been shown to be on immunoglobulin G (IgG) secreted by plasma cells. Agalactosylated IgG is positively associated with a number of cancers10,36 and systemic inflammatory states such as rheumatoid arthritis, inflammatory bowel disease,37,38 vasculitis,39 and chronic liver fibrosis.40 When reviewing the literature, striking similarities in the serum N-glycome are observed in patients with malignant and nonmalignant inflammatory diseases. For instance, acute pancreatitis previously has been shown to alter the N-glycome and increase the ratio of outer arm to core fucose, SLeX, branching and highly sialylated structures.41 Another study on

asialylated, and nonfucosylated structure (GP 1) in serum prior to surgery predict poor outcome in our cohort. Alteration of certain N-glycan structures in malignant transformation of a tumor is a well-described feature of carcinogenesis.31 One of the most characterized examples of this is the facilitation of tumor cells to increase expression of enzymes giving rise to an N-linked epitope called Sialyl-LewisX (SLeX). This epitope, present on tumor cell proteins, may aid in the extravasation of malignant cells into the circulation.9 The relationship between a localized malignant solid tumor process and the serum N-glycome is, however, more unclear. Some 20 proteins within plasma constitute of about 99% of the protein content in plasma, where the majority originate from the liver and plasma cells. Proteins shed from other tissues account for a very small fraction. Hence, there is a considerable dynamic range of concentrations for circulating proteins, likely to span more than 10 orders of magnitude.32,33 As a consequence, changes in N-linked glycans in serum may be equally marked or vague depending on whether the parent proteins are derived from the liver, plasma cells, or a solid tumor. Our data do not support any difference in the serum Nglycome of patients with resectable malignant and benign periampullary tumors. Furthermore, little association is seen between histopathology of the malignant lesions and serum Nglycan levels. The lack of association with tumor-specific characteristics have been published previously.26 The serum Nglycome is, however, highly correlated with a systemic acute 5152


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present in serum. Highly sialylated and branched glycans and glycans with SLeX produced on liver acute phase glycoproteins stay longer in circulation and have anti-inflammatory properties, and they may therefore affect cancer spread and metastasis.51 Agalactosylated glycoforms of IgG (lower galactosylation and sialylation) have increased complementdependent cytotoxicity, alter the immune response in cancer patients,40 and increase with cancer progression.9 There is evidence that certain glycoforms present in serum may also be derived from tumor cells, as with RNase 1 and PSA for pancreatic and prostate cancer, respectively, but in far lesser amounts.52,53 An elevated CRP previously has been associated with poor prognosis in patients with metastatic pancreatic cancer,54,55 whereas a prognostic role of CRP has not been established in patients with nonmetastatic disease.56 The apparent link between complex N-glycan structures and clinical outcome found by Nouso et al. could simply be reflecting similar processes as CRP in metastatic patients since the study has not stratified for the acute phase response. Nonmetastatic pancreatic disease is different, with systemic responses usually less perturbed. In the current study, we report a shift in the Nglycome with more multiantennary complex structures with both benign and malignant pancreatic lesions, clearly correlated with the acute phase response. None of these complex structures is, however, significantly associated with clinical outcome in our cohort of patients with nonmetastatic periampullary adenocarcinoma. Neither do we observe any clear association between preoperative hsCRP levels and clinical outcome. The monoantennary structure GP 1 is independent of hsCRP and is associated with poor survival in our study. This could potentially be a prognostic marker reflecting modified phenotypes of the host and/or tumor microenvironment. A few limitations of this study have to be noted. A glycosylation analysis of individual serum glycoproteins, such as IgG, has not yet been attempted on the sample set described here. Moreover, this is a retrospective case-control study, which may introduce several biases. Blood sampling may differ slightly between the cohorts. This may introduce a bias since hospitalization may trigger stress responses that the healthy controls do not display. We can therefore not exclude the possibility that some of the differences seen between the patients and healthy controls are related to other factors than tumorigenesis. Furthermore, clinical data such as significant comorbidities and medication have been recorded retrospectively based on patient journals, and data records may be incomplete. The sizes of the different groups (i.e., malignant, benign and controls) are different, and this is mainly due to the fact that the biobank has a limited number of patients with benign lesion undergoing pancreatoduodenectomy. There is a weak tendency of increased serum N-glycan branching, sialylation, galactosylation, and outer arm fucose in patients with malignant compared to benign periampullary tumors (Figure 3). This trend does not reach statistical significance, which could be due to low sampling size. There is a risk of false negative results, and studies on larger cohorts could confirm if this is a real trend. Many factors unrelated to tumorigenesis may affect the serum N-glycome including diet, smoking, medication, other diseases, and body mass index (BMI).23,57,58 Cases and controls were matched as closely as possible with regard to age, gender, and BMI. The controls are somewhat younger than the cases,

inflammatory arthritis demonstrated increased amounts of agalactosylated structures on IgG and highly branched structures with and without outer arm fucose on other serum glycoproteins.12 In vitro studies suggest that acute inflammation is associated with biantennary structures, whereas there is a shift toward tri- and tetraantennary structures in chronic inflammation and cancer.9 Surprisingly, most of the changes seen in the serum Nglycome for many solid cancers and inflammatory states seem to relate to alterations in acute phase proteins and immunoglobulins. On the other hand, there are no alterations of the serum N-glycome in malignant melanoma where inflammation plays an inferior role in early disease stages.10 It is therefore plausible that the main alterations seen in serum from pancreatic cancer patients relate to liver and plasma cell proteins rather than tumor related proteins. Our study supports the hypothesis that the observed differences in the serum N-glycome are at least partly dependent on certain immunologic triggers localized within the tumor and its microenvironment. A gene enrichment study based on whole genome expression data from patients with periampullary adenocarcinoma demonstrates a striking enrichment of networks related to local immune responses and cytokine pathways. These pathways are positively correlated with highly branched and decorated glycans with or without outer fucose. What is also interesting to see is that there are certain enriched pathways relating to extracellular matrix receptor interaction and focal adhesion, which correlate with the serum N-glycome (Figure 6). There is evidence to support that the immune system plays an important role of pancreatic adenocarcinoma development. It is suggested that tumors of the pancreas dysbalance the immune system response, thus facilitating spontaneous cancer development. The protumor cytokines (IL-1β, IL-6, IL-8, TNFα) and antitumor cytokines (IL-12, INF-γ) are examples of potential autocrine and paracrine players in the tumor microenvironment, produced by immune and cancer cells.42 It is also known that the same cytokines may act in an endocrine fashion and stimulate the acute phase response of the liver.43 One study by Okuyama and colleagues reports that hepatocytes incubated in conditioned medium from a pancreatic cancer cell line change their glycome expression,34 pointing that certain factors may stimulate hepatocytes to alter their proteoglycan composition. In vitro experiments with the hepatic cell line HuH-7 exposed to IL-1β and IL-6 demonstrated an induced expression of ST3GAL4 and FUT6, leading to a more than two-fold increase in SLeX epitope formation.44,45 The same enzymes may be induced by TNF-α via an NFκB-p65 dependent transcriptional regulation.46 Inflammatory cytokines were shown to induce increase in branching, sialylation, and SLeX epitopes,9,47 and presence of cancer with pro-inflammatory stimuli caused increase in branching and sialylation on serum liver glycoproteins in mouse model.48 Anti-TNF therapy in rheumatoid arthritis led to decreased sialylation on serum glycoproteins and increased galactosylation and sialylation on IgG.12,49 We suggest that there may be certain immunologic triggers secreted by the tumor microenvironment, as suggested by our analysis and previous reports,9,50 affecting the serum Nglycome. What exactly these factors are remains to be elucidated. It is, however, reasonable to believe that the liver and plasma cells are important targets of these factors (Figure 7) and that these tissues contribute to the bulk of glycoforms 5153


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Journal of Proteome Research but the effect of this age difference is probably minor and mostly related to GP 19. BMI levels in our study are comparable between the different groups. There is a slight difference in the number of patients presenting with any other current disease, any medication, and current smoking between the malignant and benign group (Table 2). We believe the method used is robust to screen for global Nglycan perturbations in serum, and major differences will be apparent as seen in our study. In many respects, one gets a global view of the N-glycan changes. However, there is a risk that more subtle and important alterations could be missed. It is also known that there is a higher analytical variability in the small chromatography peaks compared to the larger peaks when using this method, which makes quantification of small peaks more challenging. Still, the coefficient of variation for GP 1 is only 11.8%, which is considered acceptable.20 Finally, we have not evaluated the relationship between N-glycans and specific serum proteins, which could be useful to assess the functional role of identified alterations.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +47 22 78 13 77. Fax: +47 22 78 13 95. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank Dr. Lotte Lauritzen and Prof. Anne-Lise Børresen-Dale for contributing with healthy control samples and Prof. Kjell M. Tveit for financial support. R.S. acknowledges funding from the European Union Seventh Framework Programme (FP7/2007-2013) under Grant Agreement No. 260600 (“GlycoHIT”) and funding from Science Foundation Ireland (Starting Investigator Research Grant ID 13/SIRG/2164). E.H.K. acknowledges funding from Ivar, Ragna, and Morten Hole’s Foundation. The study was partly supported by the Research Council of Norway through its Centers of Excellence funding scheme, Project No. 179571. The authors would also like to thank Martina L. Skrede for contributing to the hsCRP analyses and Dr. Else Marit Inderberg Suso for critically reviewing the manuscript. Finally, we would like to thank all the patients that participated in the study.

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CONCLUSIONS In this study, we observed significant changes in the serum Nglycome of patients with periampullary adenocarcinoma compared to healthy individuals. We did not see any significant differences between patients with benign and malignant periampullary tumors. Many alterations in the N-glycome correlated with systemic acute phase response as measured by hsCRP. Enrichment analysis indicated that immunologic pathways of the tumors correlate with specific features of the serum N-glycome. The current study supports the hypothesis that certain factors secreted by the tumor affect liver and plasma cells to orchestrate changes in the serum N-glycome. The serum N-glycome could potentially reflect modified phenotypes of the host and/or tumor microenvironment. The prognostic impact of the serum N-glycome should be evaluated in larger, prospective studies.

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Selected R-scripts used for statistical and bioinformatical analyses (PDF)

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jproteome.5b00395. Example of a representative serum sample HILIC-UPLC chromatogram from a healthy control and a patient with periampullary adenocarcinoma; major glycan structures in each GP are highlighted; GU, glucose unit (PDF) Box plots of log 10 transformed GP levels in serum from patients with periampullary adenocarcinoma (malignant) compared to that of healthy individuals (CTR); the figure displays only significant results (p < 0.001); the highlighted plots indicate the 13 GPs that are needed to lower the misclassification error (to an acceptable level of 1%) of cancer patient versus healthy individual according to the PAM; note that there is a scale variation between each figure panel (PDF) Results from KEGG pathway enrichment analysis of correlation between tumor mRNA expression and serum N-glycan peaks for patients with periampullary adenocarcinoma (PDF) List of N-glycan structures represented in each peak and estimated features including highlighted glycan structure (PDF) 5154


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