N-glycan signatures identified in tumor interstitial fluid and serum of breast cancer patients: association with tumor biology and clinical outcome

Abstract

Particular N-glycan structures are known to be associated with breast malignancies by coordinating various regulatory events within the tumor and corresponding microenvironment, thus implying that N-glycan patterns may be used for cancer stratification and as predictive or prognostic biomarkers. However, the association between N-glycans secreted by breast tumor and corresponding clinical relevance remain to be elucidated. We profiled N-glycans by HILIC UPLC across a discovery dataset composed of tumor interstitial fluids (TIF, n = 85), paired normal interstitial fluids (NIF, n = 54) and serum samples (n = 28) followed by independent evaluation, with the ultimate goal of identifying tumor-related N-glycan patterns in blood of patients with breast cancer. The segregation of N-linked oligosaccharides revealed 33 compositions, which exhibited differential abundances between TIF and NIF. TIFs were depleted of bisecting N-glycans, which are known to play essential roles in tumor suppression. An increased level of simple high mannose N-glycans in TIF strongly correlated with the presence of tumor infiltrating lymphocytes within tumor. At the same time, a low level of highly complex N-glycans in TIF inversely correlated with the presence of infiltrating lymphocytes within tumor. Survival analysis showed that patients exhibiting increased TIF abundance of GP24 had better outcomes, whereas low levels of GP10, GP23, GP38, and coreF were associated with poor prognosis. Levels of GP1, GP8, GP9, GP14, GP23, GP28, GP37, GP38, and coreF were significantly correlated between TIF and paired serum samples. Cross-validation analysis using an independent serum dataset supported the observed correlation between TIF and serum, for five of nine N-glycan groups: GP8, GP9, GP14, GP23, and coreF. Collectively, our results imply that profiling of N-glycans from proximal breast tumor fluids is a promising strategy for determining tumor-derived glyco-signature(s) in the blood. N-glycans structures validated in our study may serve as novel biomarkers to improve the diagnostic and prognostic stratification of patients with breast cancer.

Keywords: N-glycan; biomarker; blood; heterogeneity; tumor infiltrating lymphocytes; tumor microenvironment.

Figure 1

The experimental workflow including final number of samples used in each analysis.

Figure 2

N‐glycans with differential abundance between TIF and NIF. (A) Multidimensional scaling plot. M1 and M2 are the scaling components that best captured the distance (squared euclidian) relationship between samples. Gray points denote NIF samples; colored points are TIFs, stratifying into BC subtypes luminal A (LumA), luminal B (LumB), luminal B HER2‐enriched (LumB HER2‐enriched), HER2, and TNBC. Ellipses capture the majority of samples within a single group. (B) Bar plot shows 33 N‐glycan groups with differential abundance between TIF and NIF. Height and directionality of bars indicate the log‐fold change; color depicts the scaled inverse FDR: Darker shade indicates a lower FDR. All glycans depicted in the plot had FDR ≤ 0.05. Stars mark peaks containing bisecting N‐glycans.

Figure 3

N‐glycans with differential abundance in TIL‐enriched and TIL‐depleted samples. (A) Bar plot shows N‐glycan groups with differential abundance in tumor samples with low (0/+1) and high (+2/+3) overall TIL status, as determined by CD45 positivity (see Table S2 for details). (B) Bar plot shows N‐glycan groups with differential abundance in samples with low vs. high TIL status, as determined by CD4 positivity. Height and directionality of bars indicate log‐fold change. Shade depicts inverse FDR: Darker shade indicates lower FDR. All N‐glycans depicted in the plot had FDR ≤ 0.05.

Figure 4

Prognostic association of TIF N‐glycans with overall survival. (A) Cox proportional hazards regression. Dot plot shows hazard ratios and confidence intervals for each N‐glycan group. Dot size indicates the inverse FDR, that is, small FDRs are depicted as large dots. Dot color indicates whether the abundance of a given N‐glycan was significantly associated with overall survival (FDR ≤ 0.05). (B) Survival curves based on low and high abundance of five N‐glycan groups associated with poorer overall survival and one N‐glycan group (GP24) found to be protective. Curves are fitted (simulated) using the age at diagnosis of 66 years (median of observed data). Curves show the probability of survival in years after diagnosis (at the age of 66) as a function of a high (orange) or low (blue) level of a given N‐glycan (minimum observed value—lower 25th percentile, and maximum observed value—upper 25th percentile for the N‐glycan in question).

Figure 5

Correlation of N‐glycan abundance in TIF and matched serum samples. (A) Dot plot shows Pearson's correlation coefficient between N‐glycan abundances in serum and TIF. Shade of dot represents inverse FDR. (B) Scatter plots of nine N‐glycan groups with abundances significantly correlated in serum and TIFs. Line color indicates directionality (red = negative correlation, blue = positive correlation). Gray shading shows the confidence of the regression line in a given area of the plot.

Figure 6

Overview of the main results obtained in the study. N‐glycan groups (1–46) and N‐glycan features. Rows = traits and columns = N‐glycans ID. Black dots denote which N‐glycan group and features were significantly associated with a given trait based on analysis described in the corresponding result sections. The prominent N‐glycan structures within a given N‐glycan peak are specified below the GP's ID.