Gene Expression Profiling and Prediction of Survival in Oropharyngeal Tumors

Background: Oropharynx squamous cell carcinoma (OPSCC) is a subtype of HNSCC, arising from the base of tongue, lingual tonsil, tonsil, oropharynx, pharynx. The majority of OPSCC positive for HPV infection is associated with better prognosis, but a fraction of them, similarly to HPV-negative ones, is resistant to therapy and has poor prognosis. A deep molecular study of OPSCC is mandatory to identify either prognostic markers or targets for therapy, in particular in patients with worse prognosis. Methods: 14 HPV-positive and 15 HPV-negative Italian OPSCC (n=29) with complete clinical information and follow-up of more than 5 years were molecularly characterized by gene expression profiling and compared to three cohorts of OPSCC extracted from public HNSCC datasets. AKR1C3 emerged as robust marker overexpressed in HPV-negative OPSCC and in HPV-positive lesions with worse prognosis. Fadu and Cal-27 OPSCC cell lines were treated with AKR1C3 inhibitors, alone or in combination with Cisplatin. Results: Gene set enrichment analysis revealed that up-regulated genes in HPV-positive samples are involved in immune system, muscle related processes, response to stimuli, actin organization, tissue development and adhesion, while down-regulated genes participated in glutathione derivative biosynthetic and xenobiotic metabolic processes, hypoxia and oxidative stress. AKR1C3, coding for an enzyme involved in chemio-radioresistance, is in the top10 genes with higher upregulation in HPV-negative samples. Pre-treatment with a selective AKR1C3 inhibitor potentiated the effect of Cisplatin in cells with higher basal level of the enzyme. Conclusions: We identified a druggable target, AKR1C3, associated with survival in subgroups of OPSCC patients either positive or negative for HPV infection and to resistance to chemo-radiotherapy in HNSCC. Pretreatment of OPSCC cell lines expressing this enzyme with a selective AKR1C3 inhibitor is able to enhance Cisplatin efficacy. Gene expression of 29 cases of squamous cell carcinomas of the oropharynx (OPSCC) were profiled with Agilent microarrays. Each sample was analyzed with a dye-swap technical replicate.

背景：口咽鳞状细胞癌（Oropharynx squamous cell carcinoma, OPSCC）是头颈部鳞状细胞癌（HNSCC）的亚型，起源于舌根部、舌扁桃体、扁桃体、口咽及咽腔。多数HPV（人乳头瘤病毒）阳性的OPSCC患者预后较好，但其中有一部分与HPV阴性患者类似，会出现治疗抵抗且预后不良。对OPSCC开展深入的分子研究，对于识别预后标志物或治疗靶点至关重要，尤其是针对预后较差的患者群体。
方法：本研究对14例HPV阳性、15例HPV阴性的意大利籍OPSCC患者（共29例）进行分子特征分析，所有患者均具备完整临床信息且随访时长超过5年。研究通过基因表达谱分析完成分子表征，并将结果与从公共HNSCC数据集提取的3个OPSCC队列进行比对。结果发现，AKR1C3作为稳定的标志物，在HPV阴性OPSCC以及预后不良的HPV阳性病变中均呈现过表达。随后采用AKR1C3抑制剂单独或联合顺铂（Cisplatin）处理Fadu与Cal-27两种OPSCC细胞系。
结果：基因集富集分析（Gene set enrichment analysis）显示，HPV阳性样本中的上调基因主要参与免疫系统、肌肉相关过程、应激反应、肌动蛋白组织、组织发育及黏附过程；而下调基因则参与谷胱甘肽衍生物生物合成、异生物质代谢过程、缺氧及氧化应激反应。编码参与放化疗抵抗相关酶的AKR1C3，位列HPV阴性样本中上调幅度最高的前10个基因之一。在该酶基础表达水平较高的细胞中，预先使用选择性AKR1C3抑制剂可增强顺铂的治疗效果。
结论：本研究鉴定出一个可靶向治疗的靶点AKR1C3，该靶点与OPSCC患者（无论HPV阳性还是阴性）的生存情况相关，且与HNSCC的放化疗抵抗有关。对表达该酶的OPSCC细胞系预先使用选择性AKR1C3抑制剂，能够增强顺铂的疗效。本研究通过安捷伦微阵列（Agilent microarrays）对29例口咽鳞状细胞癌样本的基因表达进行检测，每个样本均通过染料交换（dye-swap）技术完成了技术重复分析。