Glyco-signatures in patients with advanced lung cancer during anti-PD-1/PD-L1 immunotherapy

Abstract

Immune checkpoint inhibitors (ICIs) targeting programmed cell death 1/programmed cell death ligand-1 (PD-1/PD-L1) have significantly prolonged the survival of advanced/metastatic patients with lung cancer. However, only a small proportion of patients can benefit from ICIs, and clinical management of the treatment process remains challenging. Glycosylation has added a new dimension to advance our understanding of tumor immunity and immunotherapy. To systematically characterize anti-PD-1/PD-L1 immunotherapy-related changes in serum glycoproteins, a series of serum samples from 12 patients with metastatic lung squamous cell carcinoma (SCC) and lung adenocarcinoma (ADC), collected before and during ICIs treatment, are firstly analyzed with mass-spectrometry-based label-free quantification method. Second, a stratification analysis is performed among anti-PD-1/PD-L1 responders and non-responders, with serum levels of glycopeptides correlated with treatment response. In addition, in an independent validation cohort, a large-scale site-specific profiling strategy based on chemical labeling is employed to confirm the unusual characteristics of IgG N-glycosylation associated with anti-PD-1/PD-L1 treatment. Unbiased label-free quantitative glycoproteomics reveals serum levels’ alterations related to anti-PD-1/PD-L1 treatment in 27 out of 337 quantified glycopeptides. The intact glycopeptide EEQFN177STYR (H3N4) corresponding to IgG4 is significantly increased during anti-PD-1/PD-L1 treatment (FC=2.65, P=0.0083) and has the highest increase in anti-PD-1/PD-L1 responders (FC=5.84, P=0.0190). Quantitative glycoproteomics based on protein purification and chemical labeling confirms this observation. Furthermore, obvious associations between the two intact glycopeptides (EEQFN177STYR (H3N4) of IgG4, EEQYN227STFR (H3N4F1) of IgG3) and response to treatment are observed, which may play a guiding role in cancer immunotherapy. Our findings could benefit future clinical disease management.

1. Introduction

Lung cancer is one of the most common cancers and is considered as the leading cause of cancer-related mortality around the world [1]. Non-small cell lung cancer (NSCLC) accounts for more than 85% of lung cancers [2]. Only 25% of all patients with NSCLC survive more than 5 years after diagnosis, and only 6.9% of patients with advanced-stage NSCLC survive [3,4]. Immunotherapy has changed the current landscape of cancer treatment and shown unparalleled improvement in survival rates [5]. Now, about one-third of cancer patients are eligible for the most widely used class of immunotherapy, immune checkpoint inhibitors (ICIs) [6]. PD-1 and its ligand PD-L1, are critical molecules in the immune checkpoint pathway [7].

However, immune checkpoint inhibitors can only benefit a minority of cancer patients. Anti-PD-1 antibodies produce an objective response in approximately one-quarter to one-fifth of patients with melanoma or renal cell cancer [8]. The response rate of single-agent PD-1/PD-L1 inhibitors in unselected NSCLC patients is 25% [9]. Considering the response limitations, the efficacy of anti-PD-1/PD-L1 immunotherapy has become the focus of current research [10]. In many solid tumors, novel immune features have been found in cancer patients who are more likely to benefit from immune-checkpoint inhibition, such as enhanced immune cell infiltration and CD8+ T cell chemotaxis [11‒13]. In melanoma, non-responders to anti-PD1 therapy exhibit the characteristics of a de-differentiated gene expression pattern [14]. Furthermore, the dynamic changes of intestinal microbiota have a highly significant linkage to the efficacy of PD-1 inhibitor treatment [15]. These available data indicate that the regulation of ICIs response is multifactorial and requires further study.

Glycosylation is one of the most important post-translational modifications of proteins, which is the key determinant of protein biological functions [16]. Most serum proteins are glycosylated, and some of the glycan alterations in serum are gaining more and more attention [17]. On the one hand, as a fundamental feature of proteins, glycosylation has a wide range of clinical implications in multidisciplinary fields, including oncology [18].