Transcriptome and microbiome-immune changes across preinvasive and invasive anal cancer lesions

Anal squamous cell carcinoma (ASCC) is a rare gastrointestinal malignancy that is linked to high-risk Human papillomavirus (HPV) infection. It is often preceded by precursor lesions like Low-Grade Squamous Intraepithelial Lesions (LGSIL) and High-Grade Squamous Intraepithelial Lesions (HGSIL). The incidence of ASCC varies across populations, with heightened risk in HIV-positive individuals. In a previous study, we characterized the anal microbiome in high-risk HIV-exposed MSM and TGW subjects. We revealed oncogenic viromes and pertinent bacterial species associated with anal SILs. Our current investigation aimed to delineate transcriptomic and metatranscriptomic changes during the progression from precancerous lesions to ASCC. We collected 70 anal tissue samples across various lesion stages (LGSIL, HGSIL, and ASCC). Our metatranscriptomic analysis revealed that Fusobacterium nucleatum, F. gonidiaformans, Bacteroides fragilis, Campylobacter ureolyticus, and Cribacterium bergeronii were more prevalent in ASCC than in precancerous lesions. These bacterial species contributed with gene encoding enzymes (e.g.: Acca, glyQ, eno, pgk and por) and oncoproteins (FadA and dnaK) revealing potential new markers for diagnosis or treatment approaches. Our unsupervised transcriptome analysis identified two distinct sample clusters based on histological diagnosis, immune infiltrate, HIV and HPV status, and pathway activities such as immune activation, cell cycle, and antiviral signaling that recapitulate the natural history of anal cancer progression. Mutations were observed affecting KMT2C (30%), PIK3CA (21%), EP300 (21%) and NOTCH1 (13%) cancer driver genes among ASCC but also in precancerous lesions. Our study provides insights into the molecular mechanisms governing anal cancer progression, offering valuable information that may help to stratify HGSIL cases with low- or high-risk progression to the malignant stages. Overall design: A total of 70 anal samples were collected from patients diagnosed with different stages of anal lesions, including Low-Grade Squamous Intraepithelial Lesions (LGSIL), High-Grade Squamous Intraepithelial Lesions (HGSIL), and Anal Squamous Cell Carcinoma (ASCC).

肛门鳞状细胞癌（Anal squamous cell carcinoma, ASCC）是一类罕见的胃肠道恶性肿瘤，与高危型人乳头瘤病毒（Human papillomavirus, HPV）感染密切相关。该疾病常由低级别鳞状上皮内病变（Low-Grade Squamous Intraepithelial Lesions, LGSIL）和高级别鳞状上皮内病变（High-Grade Squamous Intraepithelial Lesions, HGSIL）等癌前病变进展而来。肛门鳞状细胞癌的发病率存在显著人群异质性，HIV阳性个体的患病风险显著升高。

既往研究中，本课题组对高危HIV暴露的男男性行为者（Men who have sex with men, MSM）及跨性别女性（Transgender women, TGW）的肛门微生物组进行了表征，揭示了与肛门鳞状上皮内病变相关的致癌病毒组及特定细菌类群。本研究旨在阐明从癌前病变进展至肛门鳞状细胞癌过程中的转录组与宏转录组变化规律。我们共收集了70份涵盖不同病变阶段的肛门组织样本，涵盖LGSIL、HGSIL及ASCC三个组别。

宏转录组分析结果显示，具核梭杆菌（Fusobacterium nucleatum）、变形梭杆菌（Fusobacterium gonidiaformans, F. gonidiaformans）、脆弱拟杆菌（Bacteroides fragilis）、解脲弯曲杆菌（Campylobacter ureolyticus）以及伯格霍里杆菌（Cribacterium bergeronii）在ASCC样本中的丰度显著高于癌前病变样本。上述细菌类群可通过编码Acca、glyQ、eno、pgk、por等酶类及FadA、dnaK等癌蛋白发挥致病作用，有望成为肛门癌诊断或治疗的潜在新型标志物。

无监督转录组分析基于组织学诊断结果、免疫浸润特征、HIV与HPV感染状态，以及免疫激活、细胞周期、抗病毒信号通路等通路活性，将所有样本划分为两个独立的样本簇，该聚类结果可反映肛门癌进展的自然病程。研究同时发现，在ASCC样本及癌前病变样本中均检测到癌症驱动基因突变，其中KMT2C（30%）、PIK3CA（21%）、EP300（21%）及NOTCH1（13%）的突变检出率较高。

本研究揭示了调控肛门癌进展的分子机制，可为高级别鳞状上皮内病变患者的风险分层提供重要参考，以区分低风险与高风险进展为恶性肿瘤的病例。

整体实验设计：本研究共纳入70份肛门组织样本，均来自确诊不同阶段肛门病变的患者，病变类型涵盖低级别鳞状上皮内病变（Low-Grade Squamous Intraepithelial Lesions, LGSIL）、高级别鳞状上皮内病变（High-Grade Squamous Intraepithelial Lesions, HGSIL）及肛门鳞状细胞癌（Anal squamous cell carcinoma, ASCC）。