Immune gene expression analysis for oral leukplakia samples. 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, p5) 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. Overall design: 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（Oral leukoplakia）是临床常见疾病，部分病例可进展为恶性肿瘤。增殖性口腔白斑（Proliferative leukoplakia, PL）是一种进展性、常呈多灶性的亚型，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）率显著更高。我们假设，相较于LL，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）；同时对PL与LL的组织样本开展免疫基因表达谱（gene expression profiling, GEP）分析（采用NanoString泛癌免疫肿瘤分析试剂盒）；此外借助多重免疫荧光与数字图像捕获技术，整合组织样本中的免疫细胞激活状态与空间分布模式，以进一步区分PL与LL。

在纳入的58例患者中（PL组29例，LL组29例），仅临床诊断为PL与无癌生存期显著缩短相关（风险比HR=11.25，P<0.05），而PD-L1评分可提示更差的无癌生存期（P<0.01）。PL患者在确诊后5年内发生恶性转化的风险较高。相较于LL组，PL组存在显著的CD8+T细胞与调节性T细胞（Treg）特征，且PD-L1相对过表达，这为采用预防性免疫疗法阻断PD-1/L1通路提供了坚实的理论依据。

整体实验设计：从每例口腔白斑（OL）标本的福尔马林固定石蜡包埋（FFPE）组织切片上，提取标记有上皮异型增生区域的穿刺芯样，采用Roche Diagnostics（美国印第安纳州印第安纳波利斯市）的High Pure FFPET RNA Isolation Kit试剂盒提取总RNA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年间首次确诊为OL的149例患者，其中78例为LL，71例为PL。在随机选取的58例具备可用（未耗竭）组织样本的患者中，预先设定的LL组（n=29）与PL组（n=29）在年龄、性别、吸烟史、口腔亚部位及病理诊断等基线特征上均保持均衡。

我们首先比较了所有LL与PL样本的免疫细胞类型RNA表达谱，并按组织异型增生程度进行分层分析；同时尝试探究哪些细胞毒性基因可解释LL与PL亚组间免疫细胞类型表达谱的差异。本研究通过计算全局显著性评分（Global significance scores, GSS），以衡量所选基因相对于LL或PL表型的整体差异表达程度，无需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的质量质控标准，可用于后续分析。