Comprehensive Immunoprofiling of High-Risk Oral Proliferative and Localized Leukoplakia

Abstract

Oral leukoplakia is common and may, in some cases, progress to carcinoma. Proliferative leukoplakia is a progressive, often multifocal subtype with a high rate of malignant transformation compared with the more common localized leukoplakia. We hypothesized that the immune microenvironment and gene expression patterns would be distinct for proliferative leukoplakia compared with localized leukoplakia. We summarize key clinicopathologic features among proliferative leukoplakia and localized leukoplakia and compare cancer-free survival (CFS) between subgroups. We analyze immunologic gene expression profiling in proliferative leukoplakia and localized leukoplakia tissue samples (NanoString PanCancer Immune Oncology Profiling). We integrate immune cell activation and spatial distribution patterns in tissue samples using multiplexed immunofluorescence and digital image capture to further define proliferative leukoplakia and localized leukoplakia. Among N = 58 patients (proliferative leukoplakia, n = 29; localized leukoplakia, n = 29), only the clinical diagnosis of proliferative leukoplakia was associated with significantly decreased CFS (HR, 11.25; P < 0.01; 5-year CFS 46.8% and 83.6% among patients with proliferative leukoplakia and localized leukoplakia, respectively). CD8+ T cells and T regulatory (Treg) were more abundant among proliferative leukoplakia samples (P < 0.01) regardless of degree of epithelial dysplasia, and often colocalized to the dysplasia–stromal interface. Gene set analysis identified granzyme M as the most differentially expressed gene favoring the proliferative leukoplakia subgroup (log2 fold change, 1.93; Padj < 0.001). Programmed death ligand 1 (PD-L1) was comparatively overexpressed among proliferative leukoplakia samples, with higher (>5) PD-L1 scores predicting worse CFS (Padj < 0.01). Proliferative leukoplakia predicts a high rate of malignant transformation within 5 years of diagnosis. Robust CD8+ T-cell and Treg signature along with relative PD-L1 overexpression compared with localized leukoplakia provides strong rationale for PD-1/PD-L1 axis blockade using preventative immunotherapy. Significance: This is the first in-depth profiling effort to immunologically characterize high-risk proliferative leukoplakia as compared with the more common localized leukoplakia. We observed a robust cytotoxic T-cell and Treg signature with relative overexpression of PD-L1 in high-risk proliferative leukoplakia providing a strong preclinical rationale for investigating PD-1/PD-L1 axis blockade in this disease as preventative immunotherapy.

Genetic Changes Driving Immunosuppressive Microenvironments in Oral Premalignancy

“…A recent comprehensive study of patient samples of leukoplakia identified that proliferative leukoplakia predicts a high rate of malignant transformation within 5 years of diagnosis. Interestingly, CD8 + T cell and Treg signatures with PD-L1 overexpression provides a justified approach to use anti-PD1 as immunoprevention approach in oral leukoplakia ( 124 ). Since these hydrogels are topically applied similar to TLR agonists used in melanoma, this platform provides an approach to incorporate additional immune agonists alone or to be used to increase the efficacy of checkpoint blockade.…”

Transcriptional analysis highlights three distinct immune profiles of high-risk oral epithelial dysplasia

“…In OPMD, the accumulation of immune cells is commonly observed in dysplastic areas (14)(15)(16)(17). Common changes reported in the immune microenvironment of OPMD are similar to other premalignant lesions, including CD3 lymphocytes, CD8 T cells, T regulatory (Treg) cells, M1/M2macrophages and the PD-1/PD-L1 axis (12,13,(17)(18)(19)(20)(21)(22)(23)(24)(25)(26). However, knowledge on the immune profile of OPMD remains limited.…”

Spatial PD‐L1, immune‐cell microenvironment, and genomic copy‐number alteration patterns and drivers of invasive‐disease transition in prospective oral precancer cohort

“…33 A recent spatial immune analysis in patients with a clinically distinct, extremely high-risk oral proliferative leukoplakia also observed a strong correlation of high PD-L1 scores predicting worse OCFS. 34 Although not the central driver in mediation analysis of LOH and dysplasia (Tables S1 and S2), our data suggest that PD-1/PD-L1-mediated immune evasion contributes, to some extent, to the invasive-disease transition in progression of oral oncogenesis, and therefore, provides strong rationale for PD-1/PD-L1 axis blockade in high-risk PD-L1-positive patient subgroups.…”

“…Our results are consistent with two recent, small, OPC, retrospective reports of higher cancer risk: in a study of eight patients (out of 120) with cancer, PD‐L1 expression (scored qualitatively, intensity = 2 vs. 0, 1) was found to be a predictor of OPC OCFS; 16 and in a recent cross‐sectional, case‐control study of 39 total patients, of which 19 were cases with cancer, increased PD‐L1 expression was observed in progressing versus non‐progressing dysplasias 33 . A recent spatial immune analysis in patients with a clinically distinct, extremely high‐risk oral proliferative leukoplakia also observed a strong correlation of high PD‐L1 scores predicting worse OCFS 34 . Although not the central driver in mediation analysis of LOH and dysplasia (Tables S1 and S2), our data suggest that PD‐1/PD‐L1‐mediated immune evasion contributes, to some extent, to the invasive‐disease transition in progression of oral oncogenesis, and therefore, provides strong rationale for PD‐1/PD‐L1 axis blockade in high‐risk PD‐L1–positive patient subgroups.…”