Detection of Colorectal Cancer Adaptive Mutability May Justify Combination of Targeted- and Immune-Therapies

项目摘要/摘要 结直肠癌(CRC)是癌症死亡的主要原因，如果及早发现是可以治愈的。不幸的是， 许多结直肠癌在较晚期被诊断出来，治愈率仍然很低，五年内治愈率为20% 生死存亡。当EGFR/RAS信号通路通过突变被结构性激活时，它是结直肠癌的强大驱动因素 (KRAS~40%，BRAF~10%，NRAS~5%)，但单独使用时靶向分子抑制剂(EGFRi，BRAFi， Meki或Erki)，都产生了令人失望的生存福利。到目前为止，仍然难以确定 将从EGFR/RAS通路靶向治疗中有意义受益的患者，可能是由于复杂的 抗性机制。一种普遍的观点是，对靶向治疗的抵抗是由于预先存在的 罕见的耐药突变细胞(亚克隆)，在最初的成功后扩大并重新填充肿瘤 治疗会导致永久性的克隆性耐药性。然而，最近，循环冗余码的自适应可变性(AM)在 对靶向治疗的反应已被报道为一种新的、早期的耐药机制。这一假设 提出阻断关键的、硬连接的信号通路会引起一种“应激”反应，其特征是 基因毒性、活性氧物种(ROS)和DNA错配修复酶的抑制。这些早期的 药物诱导的变化有效地导致一种亚克隆的、高度可变的状态，从而促进最终的出现 永久耐药的克隆人。微卫星不稳定性(MSI)和其他突变状态(即极点突变 肿瘤)现在已知提供更大的检查点抑制反应的可能性。因此，我们假设 由EGFR/RAS途径抑制剂诱导的AM可能导致快速、亚克隆、增强的突变性和 较高的新抗原负荷会产生一种“MSI样”状态。这种高度易变性，如果可以检测到的话，将预示着 对检查点抑制剂有反应的可能性就越高。传统的肿瘤突变负荷分析(TMB) 根据NGS的评估，癌症基因小组不太可能检测到全基因组的亚克隆TMB(ScTMB)。 标准的TMB分析检测到大量的、克隆性扩展的突变固定在肿瘤中，从它的时间 开国大业。相反，我们认为突变是由药物诱导的突变子表型引起的 很可能出现在低于检测极限的低变异等位基因频率(即亚克隆) 标准的NGS方法。在这个提案中，我们将测试scTMB发生在对 靶向治疗，并可使用超灵敏和高度特异的全基因组双链DNA进行检测 测序分析作为靶向治疗诱导突变状态的指示物(生物标记物)。我们建议 ScTMB的存在可以预测对检查点抑制剂的反应，并提示靶向治疗的好处 治疗与检查点抑制剂相结合。这些研究将带来更好的分子理解 新发现的“适应性突变”抗性机制，并有可能为 在生物标记物选择的患者亚群中使用靶向和免疫联合治疗以改善 回应。

PROJECT SUMMARY/ABSTRACT Colorectal cancer (CRC) is a major cause of cancer deaths which is curable if detected early. Unfortunately, many CRC tumors are diagnosed at more advanced stages where cure rates remain low with 20% five-year survival. The EGFR/RAS signaling pathway is a strong driver of CRC when constitutively activated via mutations (KRAS ~40%, BRAF ~10%, NRAS ~5%), but targeted molecular inhibitors, when used alone (EGFRi, BRAFi, MEKi or ERKi), have yielded disappointing survival benefit. To date, it remains difficult to identify the subset of patients who will meaningfully benefit from EGFR/RAS pathway-targeted therapies, likely due to complex resistance mechanisms. A widely held view is that resistance to targeted therapies is due to pre-existence of rare, drug-resistant mutant cells (subclones) that expand and repopulate the tumor following initial successful treatment leading to permanent, clonal resistance. Recently, however, adaptive mutability (AM) of CRC in response to targeted therapies has been reported as a novel, early resistance mechanism. This hypothesis proposes that blocking critical, hard-wired signaling pathways causes a “stress” reaction characterized by the of genotoxic, reactive oxygen species (ROS) and the suppression of DNA mismatch repair enzymes. These early drug-induced changes effectively cause a sub-clonal, hypermutable state that facilitates the eventual emergence of permanently drug-resistant clones. Microsatellite instability (MSI) and other mutator states (i.e. POLE-mutated tumors) are now known to provide a greater probability of checkpoint inhibitor responses. Thus, we hypothesize that the AM induced by EGFR/RAS pathway inhibitors may lead to rapid, sub-clonal, enhanced mutability, and a higher load of neoantigens producing an “MSI-like” state. This hypermutability, if detectable, would portend the higher likelihood of response to checkpoint inhibitors. Traditional analysis of tumor mutation burden (TMB) by NGS assessment of a cancer gene panel would not likely detect genome-wide sub-clonal TMB (scTMB). Standard TMB assays detect bountiful, clonally-expanded mutations fixed in the tumor from the time of its founding. Conversely, we propose that the mutations resulting from a rapid, drug-induced mutator phenotype will likely be present at low variant allele frequencies (i.e. subclonal) that are below the limit of detection of standard NGS approaches. In this proposal, we will test the hypothesis that scTMB occurs in response to targeted therapies, and can be detected using an ultrasensitive and highly specific genome-wide Duplex DNA sequencing assay as an indicator (biomarker) of a mutator state induced by targeted therapies. We propose that the presence of scTMB would predict responses to checkpoint inhibitors, and suggest a benefit to targeted therapies combined with checkpoint inhibitors. These studies will lead to a better molecular understanding of the newly identified “adaptive mutability” resistance mechanism, and potentially provide a solid rationale for the use of combination targeted- and immune-therapies in biomarker-selected patient subpopulations to improve response.