Development and Validation of Liquid Biopsy ddPCR and Next Generation Sequencing Assays for Diffuse Midline Glioma, K27M Variant

项目总结/摘要 弥漫性内在脑桥胶质瘤（DIPG），在美国每年影响估计200-300名儿童， 是最致命的癌症之一患有DIPG的儿童的中位生存期不到一年 这一点在三十年里没有改变。DIPG的诊断通常使用 临床症状和磁共振（MR）扫描。因为肿瘤的位置在 脑干，诊断性活检通常是避免的，虽然MR引导，立体定向针活检， 在一些专门的中心。组蛋白3基因H3.3（H3 F3 A K27 M）或H3.1中的错义突变 （HIST 1H 3B K27 M）存在于大多数DIPG中，代表早期致瘤事件。世界卫生 世界卫生组织（WHO）现在将DIPG和非脑桥中线胶质瘤一起分类为不同的病理学类型。 组称为弥漫性中线胶质瘤，H3 K27突变体（DMG）。患有H3 F3 A突变型肿瘤的儿童 与HIST 1H 3B/C突变型肿瘤相比，HIST 1H 3B/C突变型肿瘤对放疗耐受性好，复发早。H3 K27的文档 突变决定了H3-K27脱甲基酶和组蛋白脱乙酰酶临床试验使用的资格。此外，A 合作者在该提案中发表的初步研究表明，16/20（80%）的H3 K27儿童 突变肿瘤在血浆中具有可检测的循环肿瘤DNA（ctDNA）和血浆中的突变等位基因频率 与通过MR扫描测量的肿瘤体积相关，随着对治疗的初始反应而降低， 随着肿瘤进展而增加。 ResourcePath正在开发DMG-Dx，这是一种用于诊断、基因分型和 监测脑脊液和/或血浆样本中的儿童中线恶性神经胶质肿瘤，即 液体活检ddPCR检测使用先进的锁定核酸探针化学， 敏感和特异性组蛋白H3.3（H3 F3 A K27 M）或组蛋白H3.1（HIST 1H 3B/C K27 M）热点突变 定义DMG。我们的长期目标是将DMG-Dx开发为非侵入性和精确的诊断测试， 将改善临床管理和支持儿童脑干胶质瘤的药物开发。该液体 活检将满足三个未满足的需求：1）获得诊断信息，而不必进行侵入性， 风险活检仅在特殊中心可用; 2）确定临床试验的资格，3）作为 为新药临床试验提供重要客观信息的治疗反应生物标志物 包括免疫疗法。在本SBIR第I阶段，目标是：1）对DMG-Dx ddPCR进行分析验证 用于诊断的H3 K27突变的灵敏检测的测定和2）确定这些测定的可靠性， 肿瘤监测指征的定量测量。在第二阶段，我们将在临床上验证DMG-Dx， 多项临床试验的背景。DMG-Dx将通过直接向国际推广进行营销 儿科肿瘤学家和神经学家社区，他们照顾患有弥漫性中线胶质瘤的个体。

PROJECT SUMMARY/ABSTRACT Diffuse intrinsic pontine glioma (DIPG), affecting an estimated 200-300 children in the United States per year, is one of the deadliest cancers of any type. The median survival for children with DIPG is less than one year from diagnosis; which has not changed in three decades. The diagnosis of DIPG is typically made using clinical symptoms and magnetic resonance (MR) scans. Because of the location of the tumors in the brainstem, diagnostic biopsies are typically avoided, although MR-guided, stereotactic needle biopsies are available in a few specialized centers. Missense mutations in Histone 3 genes H3.3 (H3F3A K27M) or H3.1 (HIST1H3B K27M) are present in most DIPGs and represent an early tumorigenic event. The World Health Organization (WHO) now classifies DIPG and non-pontine midline gliomas together as a distinct pathologic group known as diffuse midline glioma, H3K27 mutant (DMG). Children with H3F3A mutant tumors are more resistant to radiotherapy and relapse earlier than HIST1H3B/C mutant tumors. Documentation of either H3K27 mutation determines eligibility for clinical trial use of H3-K27 demethylase and histone deacetylases. Further, a preliminary study published by collaborators in this proposal showed that 16/20 (80%) of children with H3K27 mutant tumors had detectable circulating tumor DNA (ctDNA) in plasma and mutant allele frequency in plasma correlated with tumor volume measured by MR scans, decreased with initial response to therapy, and increased upon tumor progression. ResourcePath is developing DMG-Dx, a digital droplet PCR (ddPCR) assay for diagnosis, genotyping and monitoring of midline malignant glial neoplasms of childhood in cerebrospinal fluid and/or plasma samples, i.e. liquid biopsy. The ddPCR assay uses advanced, locked-nucleic-acid probe chemistry to achieve exquisitely sensitive and specific histone H3.3 (H3F3A K27M) or histone H3.1 (HIST1H3B/C K27M) hotspot mutations which define DMG. Our long-term goal is to develop DMG-Dx as non-invasive and precise diagnostic tests that will improve clinical management and support drug development for pediatric brainstem gliomas. This liquid biopsy will serve three unmet needs: 1) obtain diagnostic information without having to perform invasive and risky biopsies available only at special centers; 2) determine eligibility for clinical trials and 3) serve as a therapeutic response biomarker providing objective information important to clinical trial of novel agents including immunotherapy. In this SBIR Phase I the aims are: 1) Perform analytic validation of DMG-Dx ddPCR assays for sensitive detection of H3K27 mutations for diagnosis and 2) Determine reliability of these assays for quantitative measurement in tumor monitoring indications. In Phase II we will clinically validate the DMG-Dx in the context of multiple clinical trials. DMG-Dx will be marketed by direct outreach to the international community of pediatric oncologists and neurologists who care for individuals with diffuse midline gliomas.