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. 2021 Oct 13;1(1):30-40.
doi: 10.1158/2767-9764.CRC-21-0060. eCollection 2021 Oct.

Comprehensive Immunoprofiling of High-Risk Oral Proliferative and Localized Leukoplakia

Glenn J Hanna  1 , Alessandro Villa  2 , Nikhil Mistry  3 , Yonghui Jia  4 , Charles T Quinn  1 , Madison M Turner  1   5 , Kristen D Felt  6 , Kathleen Pfaff  1   5 , Robert I Haddad  1 , Ravindra Uppaluri  1   7 , Scott J Rodig  1   4   5 , Sook-Bin Woo  3 , Ann Marie Egloff  4 , F Stephen Hodi  1
Affiliations

Comprehensive Immunoprofiling of High-Risk Oral Proliferative and Localized Leukoplakia

Glenn J Hanna et al. Cancer Res Commun. .

Erratum in

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; P adj < 0.001). Programmed death ligand 1 (PD-L1) was comparatively overexpressed among proliferative leukoplakia samples, with higher (>5) PD-L1 scores predicting worse CFS (P adj < 0.01). Proliferative leukoplakia predicts a high rate of malignant transformation within 5 years of diagnosis. A prominent 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 notable 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.

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Conflict of interest statement

G.J. Hanna reports grants from DFCI Medical Oncology Program during the conduct of the study, as well as other (sponsored research) from BMS, personal fees and other (sponsored research) from Exicure, grants from Gateway for Cancer Research, other (sponsored research) from Kite, other (sponsored research) from NantKwest/Altor Bioscience, personal fees and other (sponsored research) from Regeneron, personal fees and other (sponsored research) from Sanofi Genzyme, personal fees and other (sponsored research) from Bicara, personal fees from Maverick, and personal fees from Merck outside the submitted work. R.I. Haddad reports grants and personal fees from Merck, BMS, AstraZeneca, Genentech, Pfizer, Eisai, Bayer, GSK outside the submitted work. R. Uppaluri reports personal fees from Merck outside the submitted work. S.J. Rodig reports grants from Bristol Myers Squibb, grants from KITE/Gilead, grants from Merck, grants from Affimed Inc., and other from Immunitas Inc outside the submitted work. S.-B. Woo reports grants from Bristol Myers Squibb outside the submitted work. F.S. Hodi reports personal fees from Merck, Novartis, Aduro, Pionyr, Checkpoint, Surface, Compass, Torque, Rheos, Bicara, Genentech, Takeda, Eisai, Iovance, Bioentre, Gossamer, Novartis, 7Hills, Amgen, and Immunocore outside the submitted work, as well as patents for the following: MICA Related Disorders (licensed to institution per institutional policies); Tumor antigens and uses thereof (issued to institution per institutional policies); Angiopoiten-2 Biomarkers Predictive of Anti-immune checkpoint response (pending to institution per institutional policies); Compositions and Methods for Identification, Assessment, Prevention, and Treatment of Melanoma using PD-L1 Isoforms (pending to institution per institutional policies; Therapeutic peptides (issued to institution per institutional policies); Vaccine compositions and methods for restoring NKG2D pathway function against cancers (licensed to institution per institutional policies); Antibodies that bind to MHC class I polypeptide-related sequence A (licensed to to institution per institutional policies); and ANTI-GALECTIN ANTIBODY BIOMARKERS PREDICTIVE OF ANTI-IMMUNE CHECKPOINT AND ANTI-ANGIOGENESIS RESPONSES (pending to institution per institutional policies.)

Figures

FIGURE 1
FIGURE 1
Overall survival among the total population (A) and among patients with localized leukoplakia (LL; B) and proliferative leukoplakia (PL). CFS among the total population (C) and among patients with localized leukoplakia and proliferative leukoplakia (D). Kaplan–Meier method, log-rank testing. *, P < 0.05.
FIGURE 2
FIGURE 2
A, Heat map comparing RNA expression of immunoregulatory genes among localized leukoplakia (LL; top) and proliferative leukoplakia (PL; ; bottom) samples with protein expression grouped by immune cell type of importance (in each column). Cell type raw scoring (dark to light) indicates increasing absolute degree of protein RNA expression. B, Protein RNA expression of all immunomodulatory genes grouped by immune cell of interest and compared between localized leukoplakia and proliferative leukoplakia samples (Mann–Whitney test). Median immune cell expression levels compared between localized leukoplakia and proliferative leukoplakia (higher slope equating to greater difference; C) and compared by degree of epithelial dysplasia histologically (Kruskal—Wallis test; D). *, P < 0.05; two-sided.
FIGURE 3
FIGURE 3
A, Volcano plot among all cohort samples showing the log2 fold change in mRNA expression at the individual protein level plotted against adjusted −log10P value (degree of significance) among proliferative leukoplakia (PL) samples compared with localized leukoplakia (LL) samples. The top 10 genes with variable expression at higher significance are identified in blue and identified by name. *, P < 0.05; adjusted using the Benjamini–Yekutieli procedure (false discovery rate). B, Heat map of global significance scores compared among proliferative leukoplakia and localized leukoplakia (lighter color means higher positive scores) among genes/mRNA organized by immunologic function or pathway. C, Log2 fold change in mRNA expression scores among cytotoxicity genes in the immune panel among proliferative leukoplakia relative to localized leukoplakia samples. Upper and lower CI values are shown. *, P < 0.05 (adjusted) denoted by colored diamonds.
FIGURE 4
FIGURE 4
A, Box plots showing mean density (in mm2) of immune cell populations separated by localized leukoplakia (LL) and proliferative leukoplakia (PL) subgroups where each column shows individual values and mean is denoted by the solid line. Graphs plot the immune cell content within the dysplastic tissue, at the DSI, and within the stroma separately. *, P < 0.05, Mann–Whitney; two-sided. B, MIF imaging showing spatial representation of immune cells along the DSI within two cases (PL, W78, and LL, N61). DAPI, stains live cells; CYTOK, cytokeratin. C, Box plot showing mean density (in mm2) of immune cell populations separated by degree of dysplasia histologically regardless of localized leukoplakia or proliferative leukoplakia clinical phenotype. *, P < 0.05, Kruskal–Wallis; two-sided.
FIGURE 5
FIGURE 5
A, CPS and TPS characterizing PD-L1 expression compared among localized leukoplakia (LL) and proliferative leukoplakia (PL). *, P < 0.05, Mann–Whitney test; two-sided. B, PD-L1 CPS scores and mean values compared between differing regions of the DSI and within dysplastic and stromal tissues for both localized leukoplakia and proliferative leukoplakia samples. *, P < 0.05, Kruskall–Wallis test; two-sided. C, PD-L1 CPS values and mean scores compared by degree of histologic dysplasia. *, P < 0.05, Kruskall–Wallis test, two-sided. D, Kaplan–Meier estimate of CFS separated by PD-L1 CPS scores among pooled localized leukoplakia and proliferative leukoplakia samples. *, P < 0.05, log-rank testing; and bar graph showing PD-L1 CPS total counts among localized leukoplakia and proliferative leukoplakia samples. E, MIF imaging highlighting PD-L1 staining and spatial arrangement near the DSI among representative proliferative leukoplakia and localized leukoplakia samples. CYTOK, cytokeratin; DAPI, stains live cells.
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