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Abstract
Background Although immunotherapies have emerged as efficient strategies for treating cancer, biomarkers for outcome prediction are still under debate. Relevant biomarkers such as PD-L1, tumor mutational burden, and microsatellite instability (MSI) have shown limited efficacy as stand-alone markers.1 2 Moreover, aberrant glycosylation has been associated to tumor development, but its potential to predict immunotherapy outcomes has been barely explored.3 We studied the expression of glyco-immune genes looking for patterns that could provide information on glycobiological determinants associated with response to immunotherapy.
Methods TCGA data was obtained from GDC portal4 and immunotherapy-treated patients’ bulk transcriptomic data from the ENA (PRJEB23709).5 Deconvolution of the tumor microenvironment was performed using MIXTURE with LM22 signature.6 Gene signatures were obtained from MSigDB7 or literature.8 CMS and MSI classification were obtained from literature.9 Pair-wise comparisons were performed using Wilcoxon test. All data analyses were performed with R software v4.0.3.
Results TCGA-SKCM samples were clustered using glyco-immune genes, resulting in two glycoclusters (GCs). GC2 showed higher overall survival and a highly immunoactive profile (figure 1). From these profiles, we developed a Glyco-Immune Signature (GIS, 18 genes) and validated it using baseline biopsies from immunotherapy-treated patients. These patients recapitulated previously found profiles and responders were enriched in GC2 patients (figure 2). Our signature positively correlated with published response signatures and negatively correlated with resistance signatures. Finally, by building proportional-hazard models, we found that adding the GIS score to resistance scores, increased the capacity to discriminate between high and low-risk patients. To explore the potential of this GIS scores in other malignancies, we expanded our analysis to colorectal cancer. The GIS was associated with MSI status and predicted response to immunotherapy, with MSI-H patients and responders showing higher GIS score in TCGA-COAD data (figure 3). Moreover, high GIS-scoring patients presented a ‘hot’ tumor microenvironment and higher scores of immune-related signatures. Also, when analyzing the correspondence with CMS classification, CMS1 patients were high GIS-scoring patients and CMS2 patients were low GIS-scoring patients (figure 4). Interestingly, ~40% of MSI-L/MSS patients showed high GIS scores.
Conclusions Results show that this Glycoimmune signature could serve as surrogate marker for current clinical biomarkers associated with immunotherapy response.
References
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Immune infiltrate composition analysed with MIXTURE, using LM22 signature (top panel) and gene signature scores calculated as the geometric mean of the genes composing each signature (bottom panel) for TCGA-SKCM cohort samples grouped by glycocluster classification. Each value was scaled and is shown as Z-score. Top annotation shows several molecular and clinical traits for these samples: 1) barplot showing overall survival; 2) color- coded glycocluster classification; 3) color-scaled intratumoral heterogeneity (ITH), tumor mutational burden (TMB), and copy number variation load (CNV); 4) color-coded mutational status for 5 key driver genes, BRAF, KRAS, NRAS, HRAS, and NFI; 5) scatter plot showing absolute infiltrate score. Statistical significance was set to p<0.05 (*: p<0.05, **: p<0.01, ***p<0.001, ****: p<0.0001, ns: p>0.05). GC2 patients showed higher overall survival rates and a significantly higher proportion of naive and memory B cells, plasma cells, CD8 T cells, CD4 activated T cells, and M1 macrophages, with a lower proportion TMB, cytolytic score, interferon-y, and angiogenesis scores, with lower CNV and ITH.
Stacked barplots show the proportion of GC1 and GC2 patients in non-responders and responders, and boxplot show the distribution of GIS score for patients treated with immunotherapy in the validation cohort (PRJEB23709). Statistical significance was set to p<0.05 (*: p<0.05, **: p<0.01, ***: p<0.001, ****p<0.0001, ns: p>0.05).
Stacked barplots show the proportion of (A) high and low GIS-scoring patients in MSI-H, MSI-L and MSS patients, or (B) in patients predicted to respond to immunotherapy or not by the TIDE algorithm. Boxplot show the distribution of (C) GIS score by MSI status or by (D) response as predicted by TIDE algorithm. Statistical significance was set to p<0.05 (*: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001, ns: p>0.05).
Stacked barplots show the proportion of high and low GIS-scoring patients in the four CMS classes.
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