DataSheet_1_Clonal Selection of a Novel Deleterious TP53 Somatic Mutation Discovered in ctDNA of a KIT/PDGFRA Wild-Type Gastrointestinal Stromal Tumor Resistant to Imatinib.docx

The standard of care for the first-line treatment of advanced gastrointestinal stromal tumor (GIST) is represented by imatinib, which is given daily at a standard dosage until tumor progression. Resistance to imatinib commonly occurs through the clonal selection of genetic mutations in the tumor DNA, and an increase in imatinib dosage was demonstrated to be efficacious to overcome imatinib resistance. Wild-type GISTs, which do not display KIT or platelet-derived growth factor receptor alpha (PDGFRA) mutations, are usually primarily insensitive to imatinib and tend to rapidly relapse in course of treatment. Here we report the case of a 53-year-old male patient with gastric GIST who primarily did not respond to imatinib and that, despite the administration of an increased imatinib dose, led to patient death. By using a deep next-generation sequencing barcode-aware approach, we analyzed a panel of actionable cancer-related genes in the patient cfDNA to investigate somatic changes responsible for imatinib resistance. We identified, in two serial circulating tumor DNA (ctDNA) samples, a sharp increase in the allele frequency of a never described TP53 mutation (c.560-7_560-2delCTCTTAinsT) located in a splice acceptor site and responsible for a protein loss of function. The same TP53 mutation was retrospectively identified in the primary tumor by digital droplet PCR at a subclonal frequency (0.1%). The mutation was detected at a very high allelic frequency (99%) in the metastatic hepatic lesion, suggesting a rapid clonal selection of the mutation during tumor progression. Imatinib plasma concentration at steady state was above the threshold of 760 ng/ml reported in the literature for the minimum efficacious concentration. The de novo TP53 (c.560-7_560-2delCTCTTAinsT) mutation was in silico predicted to be associated with an aberrant RNA splicing and with an aggressive phenotype which might have contributed to a rapid disease spread despite the administration of an increased imatinib dosage. This result underlies the need of a better investigation upon the role of TP53 in the pathogenesis of GISTs and sustains the use of next-generation sequencing (NGS) in cfDNA for the identification of novel genetic markers in wild-type GISTs.

晚期胃肠道间质瘤（gastrointestinal stromal tumor, GIST）一线治疗的标准方案为伊马替尼，患者需每日按标准剂量持续给药直至肿瘤进展。伊马替尼耐药通常由肿瘤DNA中的基因突变克隆选择所介导，而提高伊马替尼剂量已被证实可有效克服此类耐药。不携带KIT或血小板源性生长因子受体α（platelet-derived growth factor receptor alpha, PDGFRA）突变的野生型GIST，通常对伊马替尼先天不敏感，且治疗过程中易快速复发。本文报告1例53岁胃部GIST男性患者，该患者最初对伊马替尼无应答，即便给予提高剂量的伊马替尼治疗，最终仍因疾病进展死亡。本研究采用带条形码识别的深度下一代测序分析方法，对患者的循环游离DNA（circulating cell-free DNA, cfDNA）中一组具有临床指导意义的癌症相关基因进行测序分析，以探究介导伊马替尼耐药的体细胞变异。研究人员在两份连续的循环肿瘤DNA（circulating tumor DNA, ctDNA）样本中，检测到1个此前未被报道的TP53突变（c.560-7_560-2delCTCTTAinsT），其等位基因频率显著升高；该突变位于剪接接受位点，可导致蛋白质功能丧失。通过微滴式数字聚合酶链反应（digital droplet PCR, ddPCR），研究人员在患者的原发肿瘤组织中回溯性检出该TP53突变，其亚克隆频率为0.1%。在患者的肝脏转移灶样本中，该突变的等位基因频率高达99%，提示肿瘤进展过程中该突变发生了快速的克隆选择。稳态下患者的伊马替尼血浆浓度高于文献报道的最低有效浓度阈值760 ng/ml。经计算机模拟预测，该新发TP53突变（c.560-7_560-2delCTCTTAinsT）可导致RNA剪接异常，并与肿瘤侵袭性表型相关，这或许解释了即便给予提高剂量的伊马替尼治疗，患者仍快速出现肿瘤扩散的原因。本研究结果凸显了深入探究TP53在GIST发病机制中作用的必要性，同时支持采用循环游离DNA的下一代测序技术，以鉴定野生型GIST中的新型遗传标志物。