DNMT3a Mutations Define a Pre-Leukemic Stem Cell Reservoir In Human Acute Myeloid Leukemia
DNMT3a Mutations Define a Pre-Leukemic Stem Cell Reservoir In Human Acute Myeloid Leukemia
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DNMT3a 突变定义了人类急性髓系白血病的白血病前干细胞库
DOI：
10.1182/blood.v122.21.487.487
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发表时间：
2013
期刊：
影响因子：
20.3
通讯作者：
J. Dick
中科院分区：
文献类型：
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作者：
Sasan Zandi;Amanda Mitchell;W. Chen;M. Jessica;M. Doedens;Medeiros Jessie;M. Rene;M. J. John;T. Hudson;B. Andrew;T. M. Quang;Lincoln D. Stein;J. Kennedy;M. Minden;Jean C. Y. Wang;J. Dick
人类白血病是由突变的多步累积引起的。然而，对起源细胞的身份、第一个遗传病变的性质以及随后突变的顺序仍然知之甚少，因为大多数新发急性髓性白血病 (AML) 病例是在事先没有观察到白血病前克隆扩增的情况下进行诊断的。作为检查 AML 肿瘤内遗传异质性的研究的一部分，我们对 12 名患者诊断时的样本中的 101 个常见突变白血病基因进行了深度靶向测序（读取深度 250×）。来自每个样本的正常 T 细胞在体外扩增，以提供非白血病造血组织用于比较。在 4 名患者中的 3 名中，我们意外地发现不仅在 AML 细胞中存在 DNMT3a 突变，而且在 T 细胞中也发现了低等位基因频率 (1-20%) 的 DNMT3a 突变。其他基因改变，如 NPM1 突变 (mutNPM1) 仅在 AML 细胞中发现，而在 T 细胞中未发现，这排除了污染性 AML 细胞作为培养 T 细胞中 DNMT3a 信号的来源。为了调查 T 细胞参与的发生率，通过桑格测序对诊断时 AML 患者的另外 71 个样本进行了 DNMT3a 突变筛查。 71 个 AML 样本中的 17 个 (24%) 携带 R882 密码子突变 (mutDNMT3a)，17 个样本中的 15 个 (88%) 也携带 mutNPM1。通过液滴数字 PCR (ddPCR) 测量新鲜分离的 T 细胞中的突变等位基因频率。在 17 名患者中的 12 名 (70.5%) 的 T 细胞中检测到 mutDNMT3a，但没有 mutNPM1 的证据，这表明 DNMT3a 突变发生在祖先干/祖细胞中的 NPM1 突变之前，该细胞在诊断时产生 T 细胞和显性 AML 克隆。为了直接确定是否可以在 AML 血液和骨髓样本的非白血病造血室中鉴定出携带 mutDNMT3a 等位基因的表型干细胞/祖细胞，我们对来自 10 名患者样本的高分辨表型定义的正常干细胞、祖细胞和成熟淋巴细胞部分进行了遗传分析。未检测到 mutNPM1 的 mutDNMT3a 存在于干细胞和所有下游祖细胞中，多能祖细胞 (MPP)、多淋巴祖细胞 (MLP) 和普通骨髓祖细胞 (CMP) 中的平均等位基因频率为 31.7%。 mutDNMT3a 和 mutNPM1 仅在粒细胞单核细胞祖细胞 (GMP) 和 CD33+ 母细胞中一起发现。重要的是，即使对于在成熟淋巴群体中未检测到 mutDNMT3a 的患者，在 MPP、MLP、CMP 中也发现了没有 mutNPM1 的 mutDNMT3a，这提供了强有力的证据，证明在白血病发生过程中 mutDNMT3a 先于 mutNPM1。对诊断和缓解样本的分析显示，与诊断相比，缓解时仅具有 mutDNMT3a 的细胞比例相似或更高。对来自 2 名 mutDNMT3a 和 mutNPM1 患者的诊断样本的细胞进行异种移植，主要产生非白血病多谱系移植物（19 只小鼠中的 18 只），其中大多数细胞携带 mutDNMT3a，但不携带 mutNPM1（平均等位基因频率为 57%），证实 mutDNMT3a 存在于 HSC 中。第 8 周和第 16 周的动力学分析显示，随着时间的推移，多谱系异种移植物中 mutDNMT3a 等位基因频率不断增加，表明 mutDNMT3a 比非突变 HSC 具有竞争性生长优势。总的来说，我们的结果与化疗后存活的 mutDNMT3a HSC 的 AML 患者的克隆扩增一致。因此，这些细胞可能代表了导致复发的进一步基因组进展的储存库。现在，我们的研究结果提供了在缓解期间进行治疗干预的可能性，以消除这些存活的白血病前克隆并防止大部分携带 mutDNMT3a 的 AML 患者复发。此外，我们的工作还提供了一个框架，用于识别白血病发生中的其他早期事件，并检查这些变化如何破坏正常的 HSC 功能并导致白血病。
披露：Wang：延龄草治疗/干细胞治疗：研究经费。
Leukemia in humans arises from the multistep accumulation of mutations. However, the identity of the cell of origin, the nature of the first genetic lesion and the order of subsequent mutations remain poorly understood, as most cases of de novo acute myeloid leukemia (AML) are diagnosed without prior observation of a pre-leukemic clonal expansion. As part of studies to examine intra-tumoral genetic heterogeneity in AML, we carried out deep targeted sequencing (read depth 250×) of 101 commonly mutated leukemia genes on samples from 12 patients at diagnosis. Normal T-cells from each sample were expanded in vitro to provide a non-leukemic hematopoietic tissue for comparison. In 3 of 4 patients, we unexpectedly identified DNMT3a mutation not only in AML cells but also in T-cells at a low allele frequency (1-20%). Other genetic alterations such as NPM1 mutation (mutNPM1) were found only in AML cells and not in T-cells, ruling out contaminating AML cells as the source of the DNMT3a signal in cultured T-cells. To investigate the prevalence of T cell involvement, an additional 71 samples from AML patients at diagnosis were screened by Sanger sequencing for DNMT3a mutations. 17 of 71 AML samples (24%) carried R882 codon mutations (mutDNMT3a), and 15 of 17 (88%) also carried mutNPM1. Mutant allele frequency in freshly isolated T-cells was measured by droplet digital PCR (ddPCR). mutDNMT3a with no evidence of mutNPM1 was detected in T-cells of 12 of 17 patients (70.5%), suggesting that DNMT3a mutation occurs before NPM1 mutation in an ancestral stem/progenitor cell that gives rise to both T-cells and the dominant AML clone present at diagnosis. To directly determine whether phenotypic stem/progenitor cells that carried the mutDNMT3a allele could be identified within the non-leukemic hematopoietic compartment of AML blood and bone marrow samples, we undertook genetic analysis of highly-resolved phenotypically-defined normal stem, progenitor and mature lymphoid cell fractions from 10 patient samples. mutDNMT3a without detectable mutNPM1was present in stem cells and all downstream progenitors, with mean allele frequency among multipotent progenitor (MPP), multilymphoid progenitor (MLP) and common myeloid progenitor (CMP) of 31.7%. mutDNMT3a and mutNPM1 were found together only in granulocyte monocyte progenitor (GMP) and CD33+ blasts. Importantly, even for patients in whom mutDNMT3a was not detected in mature lymphoid populations, mutDNMT3a without mutNPM1 was found in MPP, MLP, CMP, providing strong evidence that mutDNMT3a precedes mutNPM1 during leukemogenesis. Analysis of diagnostic and remission samples revealed similar or higher proportion of cells with mutDNMT3a alone at remission compared to diagnosis. Xenotransplantation of cells from the diagnostic samples of 2 patients with mutDNMT3a and mutNPM1generated predominantly non-leukemic multilineage grafts (18 of 19 mice) with predominance of cells bearing mutDNMT3a without mutNPM1 (mean allele frequency 57%), confirming that mutDNMT3a was present in HSC. Kinetic analysis at 8 and 16 weeks revealed increasing mutDNMT3a allele frequency in multilineage xenografts over time, suggesting that mutDNMT3a confers a competitive growth advantage over non-mutated HSC. Collectively, our results are consistent with the clonal expansion in AML patients of mutDNMT3a HSC that survive chemotherapy. These cells may therefore represent a reservoir for further genomic progression leading to relapse. Our findings now offer the possibility of therapeutic intervention during remission to eliminate these surviving pre-leukemic clones and prevent relapse in a large proportion of AML patients carrying mutDNMT3a. As well, our work provides a framework for the identification of other early events in leukemogenesis and examination of how these changes disrupt normal HSC function and lead to leukemia.
Disclosures: Wang: Trillium Therapeutics/Stem Cell Therapeutics: Research Funding.