N-Glycomic Profiling of Pheochromocytomas and Paragangliomas Separates Metastatic and Nonmetastatic Disease

Abstract

Context: No effective methods for separating primary pheochromocytomas and paragangliomas with metastatic potential are currently available. The identification of specific asparagine-linked glycan (N-glycan) structures, which are associated with metastasized pheochromocytomas and paragangliomas, may serve as a diagnostic tool.

Objective: To identify differences in N-glycomic profiles of primary metastasized and nonmetastasized pheochromocytomas and paragangliomas.

Setting: This study was conducted at Helsinki University Hospital, University of Helsinki, and Glykos Finland Ltd. and included 16 pheochromocytomas and paragangliomas: 8 primary metastasized pheochromocytomas or paragangliomas and 8 nonmetastasized tumors.

Methods: N-glycan structures were analyzed with matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) profiling of formalin-fixed, paraffin-embedded tissue samples.

Main outcome measure: N-glycan profile of tumor tissue.

Results: Four groups of neutral N-glycan signals were more abundant in metastasized tumors than in nonmetastasized tumors: complex-type N-glycan signals of cancer-associated terminal N-acetylglucosamine, multifucosylated glycans (complex fucosylation), hybrid-type N-glycans, and fucosylated pauci-mannose-type N-glycans. Three groups of acidic N-glycans were more abundant in metastasized tumors: multifucosylated glycans, acid ester-modified (sulfated or phosphorylated) glycans, and hybrid-type/monoantennary N-glycans. Fucosylation and complex fucosylation were significantly more abundant in metastasized paragangliomas and pheochromocytomas than in nonmetastasized tumors for individual tests but were over the false positivity critical rate, when adjusted for multiplicity testing.

Conclusions: MALDI-TOF MS profiling of primary pheochromocytomas and paragangliomas can identify diseases with metastatic potential based on their different N-glycan profiles. Thus, malignancy-linked N-glycan structures may serve as potential diagnostic tools for pheochromocytomas and paragangliomas.

Figure 1.

Exemplary MALDI-TOF mass spectra of N-glycans isolated from one metastatic and one nonmetastatic tumor: Neutral N-glycan spectra from a metastatic PGL (A, patient no. 8 in Table 1) and nonmetastatic PGL (B, patient no. 15 in Table 1), and acidic N-glycan spectra from the same metastatic (C patient no. 8 in Table 1) and nonmetastatic (D patient no. 15 in Table 1) tumors. Major and differing N-glycan signals that are discussed in the text are highlighted with schematic symbols describing putative glycan structures: blue square, N-acetyl-d-glucosamine; green circle, d-mannose; red triangle, l-fucose; yellow circle, d-galactose; purple diamond, N-acetylneuraminic acid; circled P, sulfate or phosphate ester. The glycans are detected as singly charged ions, neutral glycans as [M+Na]⁺ ions, and acidic glycans as [M-H]⁻ ions. The x-axis shows the m/z ratio. The y-axis shows the relative signal intensity in arbitrary units. The difference in the relative abundance of the glycan signal m/z 1930 may be difficult to see only by comparing the height of the signals; however, by measuring the proportion of the signal intensity of m/z 1930 from the total sum of glycan signal intensities, the difference is clear: In C, the relative abundance of the glycan signal m/z 1930 is 10.7%, whereas in D, it is 18.9%. The data are presented in numeric format in Supplemental Table 1. (E and F) Histology from the same tumors. (E) Metastatic PGL (patient no. 8 in Table 1). In the figure is one mitosis. (F) Nonmetastatic PGL (patient no. 15 in Table 1). The tumor cells have nuclear variation. Histology is a poor predictor of prognosis. Scale bar 50 µm. Magnification ×400.

Figure 2.

N-glycan profiles of metastatic and nonmetastatic PHEO and PGL tumor samples. The 40 most abundant neutral (A) and 40 acidic (B) glycan signals are shown. The height of the bar represents the average abundance of the glycan as a percentage of all detected glycans. The data are presented in numeric format in Supplemental Table 1. Four samples were analyzed in each tumor type. All glycan signals have been assigned to proposed monosaccharide compositions. Major and differing N-glycans discussed in the text are highlighted with schematic symbols describing putative glycan structures. See the legend of Fig. 1 for glycan symbols and further details. Error bars represent the standard error of the mean. The scale of the y-axis is nonlinear.

Figure 3.

PCA visualizes the differences in glycosylation of metastatic and nonmetastatic PHEO and PGL. Visualization of neutral (A) and combined neutral and acidic (B) N-glycan structural classification data as PCA results on factor planes 1 and 2. Light red, metastasized PHEO; light green, nonmetastasized PHEO; dark red, metastasized PGL; dark green, nonmetastasized PGL. Numbers refer to the patient numbering in Table 1. The percentages indicate the amount of variance captured by each of the factors (1 or 2). Dotted lines illustrate the clustering of PHEO and PGL samples in neutral glycan PCA (A) and metastasized and nonmetastasized samples in acidic and neutral glycan PCA (B). Two PGL tumors (5 and 13, marked with striped background) clustered with opposite sample group; please see Discussion in the main text.

Figure 4.

Differences of neutral (A–C) and acidic (D–E) N-glycan structural features between nonmetastasized and metastasized tumors. Distributions and means of structures shown in box and whisker plots, for neutral glycans A (hybrid type), B (fucosylation), C (complex fucosylation) and acidic glycans D (fucosylation), and E (complex fucosylation). Statistical analysis by the Mann–Whitney test reveal that individual P values are <0.05 but are over the false positivity critical rate, when adjusted for multiplicity testing by the Benjamini–Hochberg procedure. Additional statistics in Supplemental Table 4.