虽然 DNA 损伤增加是骨髓增生异常综合征 (MDS) 和急性髓性白血病 (AML) 的一个众所周知的特征，但尚不清楚骨髓的所有谱系和所有区域是否都受到同质影响。在本研究中，我们使用成熟的抗体对福尔马林固定、石蜡包埋的全切片骨髓活检进行免疫组织化学检测，以检测可识别 DNA 双链断裂的 pH2A.X（磷酸化组蛋白变体 H2A.X）。重点关注 TP53 突变和复杂核型 MDS/AML，我们发现与 TP53 野生型肿瘤病例和非肿瘤对照相比，pH2A.X DNA 损伤负担更大。为了了解 TP53 突变样本中双链断裂在谱系和空间上的变化如何，我们应用低多重免疫荧光染色和空间分析方案，通过 p53 蛋白染色和谱系标记来可视化 pH2A.X 细胞。 pH2A.X 主要标记中晚期红细胞，而早期红细胞和 CD34 母细胞相对较少。在一个典型的例子中，这些 pH2A.X 红细胞在局部组织为不同的菌落，并且每个菌落以同步水平显示 pH2A.X 斑点。这种高度协调的免疫表型表达也见于 p53 蛋白染色和推测的早期骨髓集落中。邻域聚类分析显示不同的骨髓区域在 pH2A.X /p53 红系或髓系集落中差异富集，表明 DNA 损伤反应和 p53 蛋白表达的空间异质性。 DNA损伤表型和致癌蛋白表达的谱系和结构背景与当前利用巨噬细胞吞噬作用去除白血病细胞的治疗发展相关，部分原因是不可修复的DNA损伤。 © 2024 大不列颠及爱尔兰病理学会。© 2024 大不列颠及爱尔兰病理学会。

While increased DNA damage is a well-described feature of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), it is unclear whether all lineages and all regions of the marrow are homogeneously affected. In this study, we performed immunohistochemistry on formalin-fixed, paraffin-embedded whole-section bone marrow biopsies using a well-established antibody to detect pH2A.X (phosphorylated histone variant H2A.X) that recognizes DNA double-strand breaks. Focusing on TP53-mutated and complex karyotype MDS/AML, we find a greater pH2A.X+ DNA damage burden compared to TP53 wild-type neoplastic cases and non-neoplastic controls. To understand how double-strand breaks vary between lineages and spatially in TP53-mutated specimens, we applied a low-multiplex immunofluorescence staining and spatial analysis protocol to visualize pH2A.X+ cells with p53 protein staining and lineage markers. pH2A.X marked predominantly mid- to late-stage erythroids, whereas early erythroids and CD34+ blasts were relatively spared. In a prototypical example, these pH2A.X+ erythroids were organized locally as distinct colonies, and each colony displayed pH2A.X+ puncta at a synchronous level. This highly coordinated immunophenotypic expression was also seen for p53 protein staining and among presumed early myeloid colonies. Neighborhood clustering analysis showed distinct marrow regions differentially enriched in pH2A.X+/p53+ erythroid or myeloid colonies, indicating spatial heterogeneity of DNA-damage response and p53 protein expression. The lineage and architectural context within which DNA damage phenotype and oncogenic protein are expressed is relevant to current therapeutic developments that leverage macrophage phagocytosis to remove leukemic cells in part due to irreparable DNA damage. © 2024 The Pathological Society of Great Britain and Ireland.© 2024 The Pathological Society of Great Britain and Ireland.