Immune gene expression analysis for oral leukplakia samples

Oral leukoplakia is common and may in some cases progress to carcinoma. Proliferative leukoplakia (PL) is a progressive, often multifocal subtype with a high rate of malignant transformation (MT) compared to the more common localized leukoplakia (LL). We hypothesized that the immune microenvironment and gene expression patterns would be distinct for PL compared to LL. We summarize key clinicopathologic features among PL and LL and compare cancer-free survival (CFS) between subgroups. We analyze immunologic gene expression profiling (GEP) in PL and LL 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 PL and LL. Among N=58 patients (PL: 29, LL: 29), only the clinical diagnosis of PL was associated with significantly decreased CFS (HR 11.25, p<0.01); 5-year CFS 46.8% and 83.6% among PL and LL patients, respectively. CD8+ T cells and Tregs were more abundant among PL samples (p<0.01) regardless of degree of epithelial dysplasia, and often colocalized to the dysplasia-stromal interface. Gene set analysis identified granzyme-M (GZMM) as the most differentially expressed gene favoring the PL subgroup (log2 fold change 1.93, adjusted p<0.001). PD-L1 was comparatively over-expressed among PL samples, with higher (>5) PD-L1 scores predicting worse CFS (p<0.01). PL predicts a high rate of MT within 5-years of diagnosis. Robust CD8+ T cell and Treg signature along with relative PD-L1 over-expression compared with LL provides strong rationale for PD-1/L1 axis blockade using preventative immunotherapy. RNA from each oral leukoplakia (OL) specimen was isolated from cores punched from areas of epithelial dysplasia (High Pure FFPET RNA Isolation Kit, Roche Diagnostics, Indianapolis, Indiana) marked on FFPE tissue slides and quantified. From our initial retrospective single institution cohort of 149 patients first diagnosed with an OL between 2000 and 2018, 78 had LL and 71 had PL. Among 58 randomly selected patients with available (non-exhausted) tissue samples the two prespecified groups of LL (N=29) and PL (N=29) were balanced in terms of baseline characteristics such as age, gender, smoking history, oral cavity subsite, and pathologic diagnosis. We first compared immune cell type RNA expression profiles for all LL and PL samples, and by degree of histologic atypia. We also sought to interrogate which cytotoxicity genes accounted for immune cell type profiling differences among the LL and PL subgroups. Global significance scores (GSS) were determined to measure the overall differential expression of selected genes relative to LL or PL phenotype ignoring whether genes were up- or down-regulated. Of the 58 samples, 49 passed Nanostring quality metrics for analysis.

口腔白斑病（oral leukoplakia）是一种常见疾病，部分病例可进展为癌。增殖性口腔白斑（proliferative leukoplakia, PL）是一种进展性、常为多灶性的亚型，相较于更为常见的局限性口腔白斑（localized leukoplakia, LL），其恶性转化（malignant transformation, MT）率更高。本研究假设，与LL相比，PL的免疫微环境与基因表达模式存在显著差异。本研究总结了PL与LL患者的关键临床病理特征，并比较了不同亚组的无癌生存期（cancer-free survival, CFS）。我们对PL与LL组织样本开展免疫基因表达谱（immunologic gene expression profiling, GEP）分析（采用NanoString泛癌免疫肿瘤分析试剂盒）。同时，利用多重免疫荧光染色与数字图像采集技术，整合组织样本中的免疫细胞激活与空间分布模式，以进一步区分PL与LL。
本研究共纳入58例患者（PL组29例，LL组29例），仅PL的临床诊断与显著降低的CFS相关（风险比（hazard ratio, HR）=11.25，P<0.01）；PL组与LL组患者的5年无癌生存率分别为46.8%与83.6%。PL样本中CD8+T细胞与调节性T细胞（Tregs）的丰度更高（P<0.01），且不受上皮异常增生程度影响，二者常共定位于异常增生-间质界面。基因集分析显示，颗粒酶M（granzyme-M, GZMM）是在PL亚组中表达上调最显著的差异基因（log2倍变化=1.93，校正P<0.001）。PD-L1（程序性死亡受体配体1）在PL样本中相对高表达，且PD-L1评分>5可预测更差的CFS（P<0.01）。PL提示诊断后5年内恶性转化风险较高。相较于LL，PL具有显著的CD8+T细胞与Treg特征，且PD-L1相对高表达，这为采用预防性免疫治疗靶向PD-1/PD-L1轴提供了坚实的理论依据。
本研究从福尔马林固定石蜡包埋（formalin-fixed paraffin-embedded, FFPE）组织切片上标记的上皮异常增生区域取材，通过穿刺获取组织芯，从中提取每例口腔白斑病（oral leukoplakia, OL）标本的RNA（采用罗氏诊断（Roche Diagnostics，印第安纳州印第安纳波利斯市）的High Pure FFPET RNA提取试剂盒）并进行定量。
本研究的初始回顾性单中心队列纳入2000年至2018年间首次诊断为OL的149例患者，其中78例为LL，71例为PL。在58例可获取可用（未耗竭）组织样本的随机入选患者中，LL组（N=29）与PL组（N=29）的基线特征（包括年龄、性别、吸烟史、口腔亚位点及病理诊断）均保持均衡。
本研究首先比较了所有LL与PL样本的免疫细胞类型RNA表达谱，并按组织学异型性程度进行分层分析。同时，本研究旨在明确哪些细胞毒性基因导致了LL与PL亚组间的免疫细胞类型表达谱差异。我们通过全局显著性评分（Global significance scores, GSS）来衡量相较于LL或PL表型的选定基因的整体差异表达水平，不考虑基因的上调或下调方向。在58例样本中，共有49例符合NanoString质量质控标准，可用于后续分析。