Whole exome sequencing of human tumours and cell lines to investigate how ERBB2 and KRAS Alterations mediate response to EGFR inhibitors in early stage Gallbladder Cancer

Purpose: The uncommonness of gallbladder cancer in the developed world has contributed to the generally poor understanding of the disease. The development of new and effective treatment has been and continues to be a major public health imperative. Methods: We report mutational and copy number analysis of 44 predominantly early-staged gallbladder tumors and 5-gallbladder cancer cell lines by a combination of directed and whole exome sequencing at an average coverage of 100X and above. Using gallbladder cancer cell lines and xenograft mouse models we performed phospho-proteome array profiling, followed by an in-depth functional characterization. Results: We describe recurrent activating ERBB2 somatic mutation in 6 of 44 gallbladder primary tumors with an overall mutation frequency of 13%, along with KRAS activating mutations in 3 of 44 samples. Consistent with whole exome findings, a phospho-proteomic array profile of 49-tyrosine kinase revealed constitutive phosphorylation of ERBB2 and EGFR that were found to heterodimerize. We demonstrate that treatment with ERBB2-specific, EGFR-specific shRNA or with covalent EGFR family inhibitor BIBW-2992 inhibits transformation, survival, migration, invasion, and tumor forming characteristics of gallbladder cancer cells harboring wild type or KRAS (G13D) but not KRAS (G12V) mutation. Furthermore, we show in vivo reduction in tumor size is paralleled by a reduction in the amounts of phospho-ERK in KRAS (G13D) but not in KRAS (G12V) xenografts, validating the in vitro findings Conclusion: Findings from this study implicate ERBB2 as an important therapeutic target in early stage gallbladder cancer. We also present the first evidence that the presence of KRAS (G12V), but not KRAS (G13D) mutation, may preclude gallbladder cancer patients to respond to anti-EGFR treatment, similar to the clinical algorithm commonly practiced to opt for anti-EGFR treatment in colorectal cancer.

研究背景：胆囊癌在发达国家中发病率较低，导致学界对该疾病的认知普遍不足。开发新型有效治疗手段历来且至今仍是一项重大公共健康要务。
研究方法：本研究结合定向测序与全外显子组测序（whole exome sequencing），对44例以早期分期为主的胆囊癌肿瘤组织及5株胆囊癌细胞系开展突变与拷贝数分析，测序平均覆盖度达100X及以上。我们以胆囊癌细胞系与异种移植小鼠模型为研究对象，实施磷酸化蛋白质组芯片（phospho-proteome array）谱分析，随后进行深入的功能表征。
研究结果：本研究在44例原发性胆囊癌样本中，发现6例存在反复出现的激活型ERBB2体细胞突变，总突变频率达13%；同时有3例样本携带KRAS激活突变。与全外显子组测序结果一致，对49种酪氨酸激酶的磷酸化蛋白质组芯片分析显示，ERBB2与EGFR存在组成型磷酸化，且二者可形成异二聚体。我们证实，针对ERBB2、EGFR的短发夹RNA（small hairpin RNA, shRNA）或共价EGFR家族抑制剂BIBW-2992处理，可抑制携带野生型或KRAS (G13D) 突变的胆囊癌细胞的转化、存活、迁移、侵袭及成瘤特性，但对携带KRAS (G12V) 突变的细胞无此效果。此外，我们观察到，在KRAS (G13D) 异种移植瘤模型中，肿瘤体积的减小与磷酸化ERK（phospho-ERK）水平的降低呈正相关，而在KRAS (G12V) 异种移植瘤模型中则无此现象，这验证了体外实验的结果。
研究结论：本研究结果表明，ERBB2可作为早期胆囊癌的重要治疗靶点。同时，本研究首次证实，携带KRAS (G12V) 突变（而非KRAS (G13D) 突变）可能会使胆囊癌患者无法对抗EGFR治疗产生响应，这与结直肠癌临床中常规选用抗EGFR治疗的诊疗流程一致。