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Robust and cost-effective CRISPR/Cas9 gene editing of primary tumor B cells in Eµ-TCL1 model of chronic lymphocytic leukemia 在 Eµ-TCL1 慢性淋巴细胞白血病模型中对原发肿瘤 B 细胞进行可靠且经济高效的 CRISPR/Cas9 基因编辑
IF 7.6 2区 医学 Q1 HEMATOLOGY Pub Date : 2024-08-15 DOI: 10.1002/hem3.134
Rosita Del Prete, Roberta Drago, Federica Nardi, Gaia Bartolini, Erika Bellini, Antonella De Rosa, Silvia Valensin, Anna Kabanova
<p>Ability to genetically edit primary B cells via CRISPR/Cas9 technology represents a powerful tool to study molecular mechanisms of B-cell pathogenesis. In this context, employing ribonucleoprotein complexes (RNPs), formed by recombinant Cas9 and genome-targetting single guide RNA molecules, brings in advantage of accelerated set-up and protocol robustness. Gene editing via RNP electroporation has been recently applied to primary tumor cells isolated from patients chronic lymphocytic leukemia (CLL), suggesting an efficient and valuable tool for studying leukemic cell biology and biomarker validation.<span><sup>1, 2</sup></span> The work by Nardi et al. on this topic proposed to electroporate unmanipulated primary CLL cells that are subsequently put in culture with human CD40L-expressing fibroblasts and soluble stimuli to promote CLL cell proliferation. In this context, cellular proliferation is required to achieve homozygous gene editing, whereas in unstimulated CLL cells it is possible to achieve only the heterozygous editing.<span><sup>1</sup></span> The method published by Mateos-Jaimez et al. relies on the preactivation of CLL cells with CD40L/BAFF/IL-21-expressing stromal cells, followed by RNP electroporation and continuation of the stimulatory coculture.<span><sup>2</sup></span> Both methods approach 80%–90% of editing efficiency and allow to perform downstream <i>in vitro</i> experiments on edited leukemic cells.</p><p>Application of a similar RNP-based editing approach to the widely used murine model of CLL, the Eμ-TCL1 transgenic mice,<span><sup>3</sup></span> represents a valuable and versatile tool to explore CLL biology <i>in vivo</i>. Examples illustrating its feasibility has been first shown in studies by Chakraborthy et al. and Martines et al.<span><sup>4, 5</sup></span> The published method consists in preactivating primary CD19<sup>+</sup>CD5<sup>+</sup> leukemic B cells by TLR9 agonist CpG ODN-1668, followed by RNP electroporation and intraperitoneal injection of 30 × 10<sup>6</sup> electroporated cells to promote expansion of edited leukemic cells <i>in vivo</i>. Despite this method has been proven effective, it has not been set up to expand edited TCL1 cells <i>in vitro</i>. This is associated with high experimental costs and does not allow to perform functional analysis of gene editing phenotype prior to the <i>in vivo</i> transfer, which eventually becomes not feasible if edited cells are unfit <i>in vivo</i>.</p><p>Hence, we envisioned a new approach that would allow to expand RNP-electroporated TCL1 cells <i>in vitro</i> prior to transfer. To this end, we first optimized culture conditions for TCL1 cells evaluating their viability and proliferation after treatment with different stimuli. We observed that ODN-1668 stimulation, although being efficient in activating TCL1 cells in the short-term,<span><sup>5</sup></span> does not allow to expand them <i>in vitro</i> (Supporting Information S1: Figure S1). We thus evaluated
CD40 在 CLL 增殖中心的参与被认为决定了 CLL 在体内的进展9 ,并促进对 Bcl2 抑制剂 venetoclax 等疗法的耐药性10 。我们建立了 sgRNA 组合,在体外对 Cd40 进行高效沉默,同时沉默 TCL1 细胞中预计不会产生任何表型的 Cd4 作为对照(图 2A、B)。与预期相反,我们观察到 Cd40 沉默并不影响体外 CD40 驱动的 TCL1 细胞增殖,这表明最初水平的 CD40 蛋白可能足以为细胞增殖提供能量,而在较晚的时间点,CD40 信号转导变得可有可无(图 2C)。然后,我们进行了体内竞争实验,将 Cd40 编辑和 Cd4 编辑的 TCL1 细胞注射到 C57BL/6 受体中(图 2D)。结果发现,体内Cd40沉默的肿瘤群体保持恒定水平,这反映在肿瘤群体中Cd40基因位点嵌合的恒定频率上(图2D,右图),以及与Cd4编辑细胞产生的对照肿瘤相比,CD40表达显著降低(图2E,F)。因此,我们的研究结果表明,CD40表达的缺失并不会影响白血病细胞在体内的扩增。这一结果与最近发表的一项观察结果一致,即野生型 TCL1 细胞能够在 CD40L-/- 宿主体内扩增。11 因此,通过破坏基因在肿瘤细胞或肿瘤微环境中的表达来测试基因在白血病进展中的功能的互补方法,11 有力地说明了 CD40 的功能可能会被体内 CLL 龛内的其他增殖刺激物所替代。从 sgRNA 验证步骤开始,整个基因沉默周期可在 6-8 周内完成,从而可快速评估基因在 CLL 进展中的功能。我们的方案通常能达到 80% 以上的编辑效率,并能在体外阶段对 CRISPR/Cas9 编辑进行质量控制和基因沉默的功能评估。它降低了编辑实验的成本,因为体外 TCL1 细胞扩增可使编辑过的细胞数量增加三倍,并证明通过静脉注射每只动物可有效移植低至 5 × 106 个编辑过的 TCL1 细胞。最后,我们的方法适用于体外和体内的多重基因编辑(佐证资料 S1:图 S6),在适当设置电穿孔和扩增条件后,可转化为精确的基因组编辑(Cas9 RNPs 与 DNA 模板共同电穿孔,用于同源定向修复)12-15 和其他 B 细胞类型。罗西塔-德尔普雷特(Rosita Del Prete)和罗贝塔-德拉戈(Roberta Drago)构思实验设计、进行实验、分析数据并撰写手稿;罗西塔-德尔普雷特、罗贝塔-德拉戈、费德里卡-纳尔迪、盖娅-巴托里尼、埃里卡-贝里尼、安东内拉-德罗莎和西尔维娅-瓦伦辛进行实验并分析数据;安娜-卡巴诺娃(Anna Kabanova)协调项目、指导实验设计、对获得的结果进行咨询、编辑手稿并提供资金。
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引用次数: 0
How to manage patients with germline DDX41 variants: Recommendations from the Nordic working group on germline predisposition for myeloid neoplasms 如何管理DDX41种系变异患者:北欧骨髓性肿瘤种系易感性工作组的建议。
IF 7.6 2区 医学 Q1 HEMATOLOGY Pub Date : 2024-08-13 DOI: 10.1002/hem3.145
Panagiotis Baliakas, Bianca Tesi, Jörg Cammenga, Asbjørg Stray-Pedersen, Kirsi Jahnukainen, Mette Klarskov Andersen, Helena Ågerstam, Maria Creignou, Ingunn Dybedal, Klas Raaschou-Jensen, Kirsten Grønbæk, Outi Kilpivaara, Eva Hellström Lindberg, Ulla Wartiovaara-Kautto

Increasing recognition of germline DDX41 variants in patients with hematological malignancies prompted us to provide DDX41-specific recommendations for diagnosis, surveillance, and treatment. Causative germline variants in the DDX41 predispose to the development of myeloid neoplasms (MNs), especially myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Almost 3%–5% of all patients with MDS or AML carry a pathogenic or likely pathogenic germline DDX41 variant, while half of them acquire a somatic second hit in the other allele. DDX41-associated MNs exhibit unique clinical characteristics compared to other hematological malignancies with germline predisposition: MNs occur mostly at advanced age and follow an indolent clinical course. Male carriers are more prone to develop MDS or AML than females. DDX41-associated MN is often hypoplastic, and the malignancy may be preceded by cytopenias.

血液恶性肿瘤患者中DDX41种系变异的识别率越来越高,这促使我们为诊断、监测和治疗提供针对DDX41的建议。DDX41的致病种系变异易导致骨髓性肿瘤(MNs)的发生,尤其是骨髓增生异常综合征(MDS)和急性髓性白血病(AML)。在所有 MDS 或 AML 患者中,近 3%-5% 的患者携带致病或可能致病的种系 DDX41 变体,而其中一半患者的另一个等位基因会出现体细胞二次突变。与其他具有种系易感性的血液恶性肿瘤相比,DDX41相关MN表现出独特的临床特征:多发性骨髓瘤大多发生在高龄阶段,临床症状不明显。与女性相比,男性携带者更容易罹患 MDS 或 AML。与 DDX41 相关的 MN 通常发育不良,恶性肿瘤发生前可能会出现细胞减少症。
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引用次数: 0
Don't call me “Sickler”: Confronting stigma in sickle cell disease 别叫我 "西克勒":正视镰状细胞病的耻辱。
IF 7.6 2区 医学 Q1 HEMATOLOGY Pub Date : 2024-08-13 DOI: 10.1002/hem3.137
Edeghonghon Olayemi
<p>In 2008, the World Health Organization (WHO) declared sickle cell disease (SCD) a public health problem. It is a global disease endemic in sub-Saharan Africa (SSA), where the vast majority of babies with SCD are born annually. Although SCD is considered rare in the European Union (EU), where there has been a gradual increase in prevalence, usually attributed to increased migration from parts of the world such as SSA. Advances in the medical management of SCD have led to a reduction in mortality. This reduction is skewed in favor of patients living in high-income countries, compared to those living in low- and middle-income countries where the disease is endemic. The associated organ damage as a result of increased life expectancy and the higher burden of psycho-social challenges faced daily by millions who live with SCD increases the frequency of interaction between people living with SCD and health care professionals.</p><p>The word “sickler” first appeared in the English language over four centuries ago; it was initially used to describe someone who works with a sickle. However, it came to be used to describe a person living with SCD. There is no doubt that it has become a derogatory word. People living with SCD regard being called “a sickler” as negative. They see it as a label that forces them to be seen as different or incapable, making them open to marginalization, stigmatization, and discrimination. It is unfortunate and unacceptable that healthcare workers, including specialist physicians, persist in using the derogatory word. It is still heard in the corridors, wards, and clinics of hospitals across the world. A quick literature search shows that editors and reviewers of medical publications persist in allowing its use, seemingly oblivious to the emotional pain and suffering it brings to the very people health workers are supposed to care for.</p><p>Globally, even in countries where it is endemic, SCD is poorly understood. There is a continued high prevalence of stigmatization against people living with SCD. This occurs as a result of society's misunderstanding of SCD. Health stigma has been defined as a social process with the following characteristics: exclusion, rejection, blame, and devaluation resulting from an experience or anticipation of adverse social judgment about a person or group of people who have a specific health problem.<span><sup>1</sup></span> Among people living with SCD, stigma affects every aspect of daily living and may negatively impact relationships with peers, friends, and family.<span><sup>2</sup></span></p><p>Stigmatization in SCD care is known to lead to poorer outcomes, such as being reluctant to seek in-hospital care during acute crisis events as patients, over time, develop an aversion to seeking health care services, and when they do, there is often a delay in getting attention and a general poor satisfaction with the care provided. This reluctance has been reported to include obtaining routine health
2008 年,世界卫生组织 (WHO) 宣布镰状细胞病 (SCD) 为公共卫生问题。这是一种流行于撒哈拉以南非洲(SSA)的全球性疾病,每年绝大多数患有 SCD 的婴儿都出生在撒哈拉以南非洲。虽然 SCD 在欧盟(EU)被认为是罕见病,但其发病率却在逐渐上升,这通常归因于从撒哈拉以南非洲等地移民的增加。SCD 医疗管理方面的进步已导致死亡率下降。与生活在疾病流行的中低收入国家的患者相比,生活在高收入国家的患者的死亡率有所下降。由于预期寿命的延长而导致的相关器官损伤,以及数百万 SCD 患者每天面临的更大的社会心理挑战负担,增加了 SCD 患者与医疗保健专业人员之间的互动频率。然而,后来它被用来描述 SCD 患者。毫无疑问,它已成为一个贬义词。SCD 患者认为被称为 "镰刀手 "是负面的。他们认为这是一个迫使他们被视为与众不同或没有能力的标签,使他们容易被边缘化、污名化和歧视。令人遗憾和无法接受的是,包括专科医生在内的医护人员仍在使用这个贬义词。在世界各地医院的走廊、病房和诊所里,人们仍然能听到这个词。在全球范围内,即使在 SCD 流行的国家,人们对它的了解也很少。针对 SCD 患者的污名化现象仍然十分普遍。这是因为社会对 SCD 的误解。健康污名化被定义为具有以下特征的社会过程:由于经历或预期社会对有特定健康问题的个人或群体的不利判断而产生的排斥、拒绝、指责和贬低。众所周知,SCD 护理中的污名化会导致较差的结果,例如在急性危机事件中患者不愿意寻求院内护理,因为随着时间的推移,患者会对寻求医疗保健服务产生厌恶感,而当他们寻求医疗保健服务时,往往会延迟得到关注,并且对所提供的护理普遍不满意。Galadanci 等人进行的一项研究3 表明,在疾病流行的地区,与 SCD 相关的耻辱感是寻求和使用婚前遗传咨询和筛查的障碍。据了解,SCD 患者曾因最初受到医护人员的污名化而停止治疗,以避免引起不必要的关注。同样,资深医生在病历中描述 SCD 患者时所使用的污名化语言对医科学生、住院医生和研究员等受训者对 SCD 患者的态度产生了负面影响,包括他们的处方用药行为;这一将偏见从一名临床医生传播到另一名临床医生的重要途径常常被忽视。4 一项针对急诊医生的研究显示,超过半数的受访医生经常或总是使用 "病态患者 "一词。5 在护理 SCD 患者的过程中持续使用污名化语言所产生的负面影响是非常重要的,因为在急性疼痛事件中,一些 SCD 患者在没有任何正当理由或证据的情况下不断被贴上寻求阿片类药物或阿片类药物依赖行为的标签。在全球范围内,众所周知,患者在接受医疗护理时并没有得到平等的对待,他们所接受的护理质量往往取决于他们的种族/民族身份;因此,以非洲裔为主的 SCD 患者经常受到歧视也就不足为奇了。与哮喘、糖尿病和囊性纤维化等其他慢性疾病患者相比,SCD 患者遭受的歧视和侮辱更多。
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引用次数: 0
What is lost when training goes digital? 培训数字化会带来什么损失?
IF 7.6 2区 医学 Q1 HEMATOLOGY Pub Date : 2024-08-08 DOI: 10.1002/hem3.148
Tanya Freeman, Stephen P. Hibbs
<p>Haematology professionals in training have a world of learning at their fingertips. Well-written textbooks and review articles are joined by YouTube channels, Twitter accounts, and data interpretation banks. Professional haematology organisations have invested in digital education, providing extensive repositories of podcasts, image banks, and recorded lecture series. Regional and national postgraduate haematology training days and haematology conferences can now be ‘attended’ through a laptop and an internet connection.</p><p>There are clear benefits of digital repositories and remote meeting software for both trainers and trainees. With so many resources to choose from, trainees can direct their own learning through whatever style works best for them<span><sup>1</sup></span> and learn at their own pace. Accessibility is increased: a trainer gives a single lecture for a ‘live’ group, and the recording can be used by trainees who are unwell, who work part-time, or who have clinical commitments during the learning session. Virtual and prerecorded teaching can be used at scale and reduces costs. For international or distant meetings, virtual attendance has a much smaller carbon footprint.</p><p>The COVID-19 pandemic accelerated the transition to digital and asynchronous training which may be coagulating to a ‘new normal’. In this article, we argue that this transition should be questioned by presenting five overlooked benefits of in-person, synchronous (‘live’) haematology training.</p><p>First, a face-to-face fixed training commitment provides a protected space and time and a positive social pressure to engage <i>now</i>. Training is often ‘protected’ from clinical commitments, and social etiquette requires enough trainees to be present to avoid embarrassing the trainer. In contrast, virtual sessions tempt trainees to attempt to multitask or defer the learning opportunity altogether, imagining that they can watch the recording later. However, the session recording may join the ever-increasing pile of things we might one day listen to or read—but probably won't get round to.</p><p>Second, live sessions provide a clear and immediate focus for learning. As digital educational repositories proliferate, the amount of available educational material can become overwhelming. How do you decide what to look at or listen to? Is a particular learning resource still up to date? In contrast, live sessions provide focus and a clear agenda for learning. Trainers can highlight major updates and curate the most valuable reading and educational resources to refer to before or after the live session. Interactive sessions can also reveal to trainees the topics that are ‘unknown unknowns’: gaps in their knowledge that they didn't know they needed to know.</p><p>Third, face-to-face training helps trainees to learn from each other. Interacting and connecting with others is essential for learning individually and collectively.<span><sup>2</sup></span> Different ‘learni
此外,在主要教学活动之前和之后的时间里,我们还有机会叙叙旧,处理一些临床上的小话题。这些互动是培养专业认同感和信任感的关键部分;在面对面交流时,这些互动不会引人注目。面对面培训通常比在线培训更令人愉快。当我们面对面时,我们可以享受彼此的陪伴,有人开玩笑,有人送来食物,有人煮咖啡。这种边学习边共度时光的乐趣本质上是好的,有助于支撑我们度过工作中更困难的部分。录制的讲座、图片库和虚拟会议很可能足以实现某些学习目标,尤其是在注重知识积累的情况下。数字资料库是准备研究生考试的绝佳资源,而自主学习是在整个职业生涯中培养的一项重要技能。但是,我们也需要建立和塑造专业身份、发展同事间信任以及花时间享受彼此陪伴的空间。在医学培训的历史上,面对面培训对实现这些目标至关重要,在一个日益数字化和异步化的世界里,面对面培训仍然值得保留。Tanya Freeman撰写了初稿。Stephen P. Hibbs对文章进行了严格审阅和修改。Tanya Freeman得到了皇家病理学院和巴兹健康NHS信托基金的教学奖学金支持。斯蒂芬-P.-希布斯(Stephen P. Hibbs)由惠康基金会资助的 HARP 博士研究奖学金支持(资助编号 223500/Z/21/Z)。本出版物未获得任何资助。
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引用次数: 0
Development of a natural language processing pipeline for assessment of cardiovascular risk in myeloproliferative neoplasms 开发用于评估骨髓增生性肿瘤心血管风险的自然语言处理管道。
IF 7.6 2区 医学 Q1 HEMATOLOGY Pub Date : 2024-08-08 DOI: 10.1002/hem3.143
Andrea Duminuco, Joshua Au Yeung, Raj Vaghela, Sukhraj Virdee, Claire Woodley, Susan Asirvatham, Natalia Curto-Garcia, Priya Sriskandarajah, Jennifer O'Sullivan, Hugues de Lavallade, Deepti Radia, Shahram Kordasti, Giuseppe Palumbo, Claire Harrison, Patrick Harrington
<p>A central feature of myeloproliferative neoplasms (MPN) is an increased risk of cardiovascular thrombotic complications, and this is the primary determinant for the introduction of cytoreductive therapy.<span><sup>1</sup></span> The landmark ECLAP study in polycythemia vera (PV) patients, showed cardiovascular mortality accounted for 45% of all deaths, with a thrombosis incidence rate of 1.7/100 person/year and a cumulative incidence of 4.5% over a median follow-up of 2.8 years.<span><sup>2</sup></span></p><p>Natural language processing (NLP) is a branch of machine learning involving computational interpretation and analysis of human language. CogStack (https://github.com/CogStack), is an open-source software ecosystem, that retrieves structured and unstructured components of electronic health records (EHR). The Medical Concept Annotation Toolkit (MedCAT), the NLP component of CogStack, structures clinical free text by disambiguating and capturing synonyms, acronyms, and contextual details, such as negation, subject, and grammatical tense, and mapping text to medical Systematized Nomenclature of Medicine–Clinical Terms (SNOMED-CT) concepts. This technique is known as “named entity recognition and linkage” (NER+L). MedCAT has previously been used and validated in many studies to structure EHR data across a range of medical specialties for auditing, observational studies, de-identifying patient records, operational insights, disease modeling, and prediction.<span><sup>3-8</sup></span></p><p>We employed our NLP pipeline, Cogstack, and MedCAT, to determine the prevalence and impact of cardiovascular risk factors upon thrombotic events during follow-up. We used Cogstack to retrieve outpatient hematology clinic letters and hematology discharge letters. MedCAT was then used for NER+L of relevant clinical free-text to respective SNOMED-CT codes that were determined by two hematology specialists. The base MedCAT model was trained unsupervised on >18 million EHR documents, and this was further fine-tuned using a 80:20 train:test split with 600 clinician-annotated MPN-specific documents. Total SNOMED-CT code counts were aggregated and grouped by individual patient, a unique threshold count was then applied to “infer” presence of the respective SNOMED code. In this process, hematology specialists read through clinical documents and manually highlight correct words or phrases detected by MedCAT that correspond to the SNOMED concept of interest.</p><p>We deploy a two-step validation process that has been well described.<span><sup>3, 8</sup></span> The first is to evaluate and validate the NER model performance on a document level demonstrating how accurately MedCAT is able to identify the medical concepts of interest. This involves hematology specialists annotating medical concepts and comparing this to the model NER outputs (Supporting Information S1: Table 1). The second step involves manual validation by creating a gold-standard real-world dataset. Tw
骨髓增生性肿瘤(MPN)的一个主要特征是心血管血栓并发症风险增加,这也是采用细胞再生疗法的主要决定因素。对多发性红细胞增多症(PV)患者进行的具有里程碑意义的 ECLAP 研究显示,心血管死亡占所有死亡的 45%,血栓形成发生率为 1.7/100人/年,中位随访 2.8 年的累积发生率为 4.5%。CogStack (https://github.com/CogStack) 是一个开源软件生态系统,可检索电子健康记录 (EHR) 中的结构化和非结构化组件。医学概念注释工具包(MedCAT)是 CogStack 的 NLP 组件,它通过消歧和捕捉同义词、缩略词和上下文细节(如否定词、主语和语法时态),将文本映射到医学术语系统化命名法(SNOMED-CT)概念,从而构建临床自由文本。这种技术被称为 "命名实体识别和链接"(NER+L)。MedCAT 此前已在多项研究中得到应用和验证,可用于构建一系列医疗专科的电子病历数据,以进行审计、观察研究、去身份化患者记录、业务洞察、疾病建模和预测3-8。我们使用 Cogstack 来检索血液科门诊信件和血液科出院信件。然后使用 MedCAT 对相关的临床自由文本进行 NER+L 处理,使之与两位血液学专家确定的 SNOMED-CT 代码相对应。基础 MedCAT 模型在 1800 万份电子病历文档上进行了无监督训练,并通过 600 份临床医生注释的 MPN 特定文档,以 80:20 的训练与测试比例对其进行了进一步微调。对 SNOMED-CT 代码的总计数进行汇总,并按单个患者进行分组,然后应用独特的阈值计数来 "推断 "是否存在相应的 SNOMED 代码。在这一过程中,血液学专家通读临床文档,并手动高亮显示 MedCAT 检测到的与相关 SNOMED 概念相对应的正确单词或短语。我们采用了两步验证流程,这一流程已得到充分描述。这需要血液学专家对医学概念进行注释,并将其与 NER 模型的输出结果进行比较(佐证资料 S1:表 1)。第二步是通过创建黄金标准真实世界数据集进行人工验证。随机指派两名血液学专家审阅患者样本临床笔记的子样本(ET 队列中的样本数为 112 [20%],PV 队列中的样本数为 60 [17%]),并结合整个临床病史,说明患者是否存在所选的 SNOMED 概念(佐证资料 S1:表 2 和表 3)。最后,利用人工验证的数据集,使用阈值优化器为真实世界的 F1 推断找到最佳概念数。人工验证至关重要,因为虽然 ML 模型可能会在文档层面上错误标注概念,但关键因素是模型在真实世界中识别患者层面上临床实体存在与否的性能如何。所选的 SNOMED 概念是心血管风险因素,包括高血压 (HTN)、高胆固醇血症 (HC)、糖尿病 (DM)、吸烟状态和肥胖。我们还对心血管事件进行了评估,包括门静脉血栓 (PVT)、深静脉血栓 (DVT)、肺栓塞 (PE)、心肌梗塞 (MI)、中风/脑血管意外 (CVA)、脑窦血栓 (CST) 和未注明的血栓 (NOS)。对 2005 年 1 月至 2023 年 4 月期间在 Guys' and St Thomas NHS Foundation Trust (GSTT) 至少就诊过一次的 360 名 PV 和 560 名 ET 患者的数据进行了评估(佐证资料 S1:表 4)。共审查了 560 名 ET 患者的 12905 份文件(中位数为每名患者 20 份,四分位数间距 [IQR],8-34)和 360 名 PV 患者的 11250 份文件(中位数为每名患者 27 份,四分位数间距 [IQR],11-47)。在人工验证数据集(ET 患者人数为 112 人,PV 患者人数为 60 人)中,MedCAT 在特异性和灵敏度方面取得了极佳的真实世界 F1 分数(佐证资料 S1:表 2 和表 3)。在 ET 队列中,使用阈值&gt;2 次提及来定义阳性人群,21.3%(119 例)的患者发现了高血压,4.6%(26 例)的患者发现了糖尿病,3.6%(20 例)的患者发现了心肌梗塞,7.7%(43 例)的患者发现了 CVA,8%(45 例)的患者发现了 NOS 血栓,1.4%(8 例)的患者发现了深静脉血栓,1.4%(8 例)的患者发现了 PE。 Hugues de Lavallade 曾获得 Incyte 的研究基金和酬金,以及诺华和辉瑞的酬金。克莱尔-哈里森(Claire Harrison)从诺华、杨森、CTI、Celgene、Medscape 等公司获得演讲酬金,并担任过 Incyte、CTI、Sierra Oncology、诺华、Celgene、罗氏、AOP pharma、Geron 和 Astra Zenica 的顾问委员会成员,还是 HemaSphere 的编辑。Patrick Harrington 从葛兰素史克、BMS、诺华、Incyte、AOP 和 Constellation 获得研究经费,并从葛兰素史克、Incyte 和诺华获得酬金。
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引用次数: 0
PD-L1+ macrophage and tumor cell abundance and proximity to T cells in the pretreatment large B-cell lymphoma microenvironment impact CD19 CAR-T cell immunotherapy efficacy 大 B 细胞淋巴瘤预处理微环境中 PD-L1+ 巨噬细胞和肿瘤细胞的丰度以及与 T 细胞的接近程度影响 CD19 CAR-T 细胞免疫疗法的疗效。
IF 7.6 2区 医学 Q1 HEMATOLOGY Pub Date : 2024-08-07 DOI: 10.1002/hem3.142
Alexandre V. Hirayama, Jocelyn H. Wright, Kimberly S. Smythe, Salvatore Fiorenza, Akira N. Shaw, Jordan Gauthier, David G. Maloney, Kikkeri N. Naresh, Cecilia C. S. Yeung, Cameron J. Turtle

CD19-targeted chimeric antigen receptor T-cell (CAR-T) immunotherapy has transformed the management of relapsed/refractory large B-cell lymphoma (LBCL), yet durable remissions are observed in less than half of treated patients. The tumor microenvironment (TME) is a key and understudied factor impacting CD19 CAR-T therapy outcomes. Using NanoString nCounter transcriptome profiling (n = 24) and multiplex immunohistochemistry (mIHC, n = 15), we studied the TME in pretreatment biopsies from patients with LBCL undergoing CD19 CAR-T therapy. Patients who achieved complete response (CR) after CAR-T therapy demonstrated higher expression of genes associated with T-cell trafficking and function, whereas those who did not achieve CR had higher expression of genes associated with macrophages and T-cell dysfunction. Distinct patterns of immune infiltration and fibrosis in the TME were associated with CAR-T therapy outcomes, and these findings were corroborated using artificial intelligence-assisted image analyses. Patients who achieved CR had a lower proportion of the biopsy occupied by an interspersed immune infiltrate and a higher proportion of hypocellular/fibrotic regions. Furthermore, mIHC revealed lower density of CD4+ T cells and higher densities of both macrophages and tumor cells expressing PD-L1 in non-CR patients. Spatial analysis revealed that PD-1+ T cells were in close proximity to PD-L1+ macrophages or PD-L1+ tumor cells in patients who did not compared to those who did achieve CR after CAR-T therapy. These findings suggest that morphologic patterns in the TME and engagement of the PD-1/PD-L1 axis in pretreatment biopsies may impact CD19 CAR-T immunotherapy response in patients with LBCL.

CD19靶向嵌合抗原受体T细胞(CAR-T)免疫疗法改变了复发/难治性大B细胞淋巴瘤(LBCL)的治疗方法,但只有不到一半的患者能获得持久缓解。肿瘤微环境(TME)是影响 CD19 CAR-T 疗法疗效的一个关键因素,但对它的研究还不够。我们使用 NanoString nCounter 转录组图谱分析(n = 24)和多重免疫组化(mIHC,n = 15)研究了接受 CD19 CAR-T 疗法的 LBCL 患者治疗前活检中的肿瘤微环境。接受CAR-T疗法后获得完全应答(CR)的患者与T细胞迁移和功能相关的基因表达较高,而未获得CR的患者与巨噬细胞和T细胞功能障碍相关的基因表达较高。TME中免疫浸润和纤维化的不同模式与CAR-T疗法的结果有关,人工智能辅助图像分析证实了这些发现。获得CR的患者活检组织中免疫浸润的比例较低,细胞减少/纤维化区域的比例较高。此外,mIHC 显示非 CR 患者的 CD4+ T 细胞密度较低,而巨噬细胞和表达 PD-L1 的肿瘤细胞密度较高。空间分析显示,与接受CAR-T疗法后达到CR的患者相比,未达到CR的患者中PD-1+ T细胞与PD-L1+巨噬细胞或PD-L1+肿瘤细胞的距离较近。这些研究结果表明,治疗前活检组织中TME的形态模式和PD-1/PD-L1轴的参与可能会影响LBCL患者对CD19 CAR-T免疫疗法的反应。
{"title":"PD-L1+ macrophage and tumor cell abundance and proximity to T cells in the pretreatment large B-cell lymphoma microenvironment impact CD19 CAR-T cell immunotherapy efficacy","authors":"Alexandre V. Hirayama,&nbsp;Jocelyn H. Wright,&nbsp;Kimberly S. Smythe,&nbsp;Salvatore Fiorenza,&nbsp;Akira N. Shaw,&nbsp;Jordan Gauthier,&nbsp;David G. Maloney,&nbsp;Kikkeri N. Naresh,&nbsp;Cecilia C. S. Yeung,&nbsp;Cameron J. Turtle","doi":"10.1002/hem3.142","DOIUrl":"10.1002/hem3.142","url":null,"abstract":"<p>CD19-targeted chimeric antigen receptor T-cell (CAR-T) immunotherapy has transformed the management of relapsed/refractory large B-cell lymphoma (LBCL), yet durable remissions are observed in less than half of treated patients. The tumor microenvironment (TME) is a key and understudied factor impacting CD19 CAR-T therapy outcomes. Using NanoString nCounter transcriptome profiling (<i>n</i> = 24) and multiplex immunohistochemistry (mIHC, <i>n</i> = 15), we studied the TME in pretreatment biopsies from patients with LBCL undergoing CD19 CAR-T therapy. Patients who achieved complete response (CR) after CAR-T therapy demonstrated higher expression of genes associated with T-cell trafficking and function, whereas those who did not achieve CR had higher expression of genes associated with macrophages and T-cell dysfunction. Distinct patterns of immune infiltration and fibrosis in the TME were associated with CAR-T therapy outcomes, and these findings were corroborated using artificial intelligence-assisted image analyses. Patients who achieved CR had a lower proportion of the biopsy occupied by an interspersed immune infiltrate and a higher proportion of hypocellular/fibrotic regions. Furthermore, mIHC revealed lower density of CD4<sup>+</sup> T cells and higher densities of both macrophages and tumor cells expressing PD-L1 in non-CR patients. Spatial analysis revealed that PD-1<sup>+</sup> T cells were in close proximity to PD-L1<sup>+</sup> macrophages or PD-L1<sup>+</sup> tumor cells in patients who did not compared to those who did achieve CR after CAR-T therapy. These findings suggest that morphologic patterns in the TME and engagement of the PD-1/PD-L1 axis in pretreatment biopsies may impact CD19 CAR-T immunotherapy response in patients with LBCL.</p>","PeriodicalId":12982,"journal":{"name":"HemaSphere","volume":"8 8","pages":""},"PeriodicalIF":7.6,"publicationDate":"2024-08-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11303978/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141901573","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
A cellular reporter system to evaluate endogenous fetal hemoglobin induction and screen for therapeutic compounds 用于评估内源性胎儿血红蛋白诱导和筛选治疗化合物的细胞报告系统。
IF 7.6 2区 医学 Q1 HEMATOLOGY Pub Date : 2024-08-06 DOI: 10.1002/hem3.139
Thijs C. J. Verheul, Nynke Gillemans, Kerstin Putzker, Rezin Majied, Tingyue Li, Memnia Vasiliou, Bert Eussen, Annelies de Klein, Wilfred F. J. van IJcken, Emile van den Akker, Marieke von Lindern, Joe Lewis, Ulrike Uhrig, Yukio Nakamura, Thamar van Dijk, Sjaak Philipsen

Reactivation of fetal hemoglobin expression alleviates the symptoms associated with β-globinopathies, severe hereditary diseases with significant global health implications due to their high morbidity and mortality rates. The symptoms emerge following the postnatal transition from fetal-to-adult hemoglobin expression. Extensive research has focused on inducing the expression of the fetal γ-globin subunit to reverse this switch and ameliorate these symptoms. Despite decades of research, only one compound, hydroxyurea, found its way to the clinic as an inducer of fetal hemoglobin. Unfortunately, its efficacy varies among patients, highlighting the need for more effective treatments. Erythroid cell lines have been instrumental in the pursuit of both pharmacological and genetic ways to reverse the postnatal hemoglobin switch. Here, we describe the first endogenously tagged fetal hemoglobin reporter cell line based on the adult erythroid progenitor cell line HUDEP2. Utilizing CRISPR-Cas9-mediated knock-in, a bioluminescent tag was integrated at the HBG1 gene. Subsequent extensive characterization confirmed that the resulting reporter cell line closely mirrors the HUDEP2 characteristics and that the cells report fetal hemoglobin induction with high sensitivity and specificity. This novel reporter cell line is therefore highly suitable for evaluating genetic and pharmacologic strategies to induce fetal hemoglobin. Furthermore, it provides an assay compatible with high-throughput drug screening, exemplified by the identification of a cluster of known fetal hemoglobin inducers in a pilot study. This new tool is made available to the research community, with the aspiration that it will accelerate the search for safer and more effective strategies to reverse the hemoglobin switch.

β-球蛋白病是一种严重的遗传性疾病,因其发病率和死亡率高而对全球健康产生重大影响。这些症状是在出生后从胎儿血红蛋白表达向成人血红蛋白表达过渡时出现的。大量研究集中于诱导胎儿γ-球蛋白亚基的表达,以逆转这种转换并改善这些症状。尽管进行了数十年的研究,但只有羟基脲一种化合物作为胎儿血红蛋白的诱导剂被应用于临床。遗憾的是,它的疗效因人而异,因此需要更有效的治疗方法。红细胞细胞系在研究逆转出生后血红蛋白转换的药物和遗传方法方面发挥了重要作用。在这里,我们描述了第一个基于成人红细胞祖细胞系 HUDEP2 的内源性标记胎儿血红蛋白报告细胞系。利用 CRISPR-Cas9 介导的基因敲入技术,在 HBG1 基因上整合了一个生物发光标签。随后进行的广泛表征证实,所得到的报告细胞系与 HUDEP2 的特征非常接近,而且细胞报告胎儿血红蛋白诱导的灵敏度和特异性都很高。因此,这种新型报告细胞系非常适合用于评估诱导胎儿血红蛋白的基因和药物策略。此外,它还提供了一种与高通量药物筛选兼容的检测方法,例如在一项试验研究中鉴定出了一组已知的胎儿血红蛋白诱导物。向研究界提供这一新工具的目的是希望它能加速寻找更安全、更有效的策略来逆转血红蛋白转换。
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引用次数: 0
The PI3Kδ inhibitor zandelisib on intermittent dosing in relapsed/refractory follicular lymphoma: Results from a global phase 2 study PI3Kδ抑制剂zandelisib在复发/难治性滤泡性淋巴瘤中的间歇用药:一项全球性2期研究的结果。
IF 7.6 2区 医学 Q1 HEMATOLOGY Pub Date : 2024-08-06 DOI: 10.1002/hem3.138
Andrew D. Zelenetz, Wojciech Jurczak, Vincent Ribrag, Kim Linton, Graham P. Collins, Javier L. Jiménez, Mark Bishton, Bhagirathbhai Dholaria, Andrea Mengarelli, Tycel J. Phillips, Nagendraprasad Sungala, Gerardo Musuraca, Oonagh Sheehy, Eric Van Den Neste, Mitsuhiko Odera, Lu Miao, Daniel P. Gold, Richard G. Ghalie, Pier L. Zinzani

In this global phase 2 study in patients with relapsed/refractory follicular lymphoma (FL), zandelisib was administered on intermittent dosing to mitigate immune-related adverse events and infections that have been reported with oral PI3Kδ inhibitors administered daily continuously. Eligible patients with measurable disease and progression after at least two prior therapies were administered zandelisib until disease progression or intolerability. The primary efficacy endpoint was objective response rate (ORR) and the key secondary efficacy endpoint was duration of response (DOR). We report on 121 patients with FL administered zandelisib on intermittent dosing after 8 weeks of daily dosing for tumor debulking. The median number of prior therapies was 3 (range, 2–8) and 45% of patients had refractory disease. The ORR was 73% (95% confidence interval [CI], 63.9–80.4), the complete response (CR) rate was 38% (95% CI, 29.3–47.3), and the median DOR was 16.4 months (95% CI, 9.5–not reached). With a median follow-up of 14.3 months (range, 1–30.5), the median progression-free survival was 11.6 months (95% CI, 8.3–not reached). Twenty-one patients (17%) discontinued therapy due to an adverse event. Grade 3–4 class-related toxicities included 6% diarrhea, 5% lung infections, 3% colitis (confirmed by biopsy or imaging), 3% rash, 2% AST elevation, and 1% non-infectious pneumonitis. Zandelisib achieved a high rate of durable responses in heavily pretreated patients with relapsed/refractory FL. The intermittent dosing resulted in a relatively low incidence of severe class-related toxicities, which supports the evaluation of zandelisib as a single agent and in combination with indolent B-cell malignancies.

在这项针对复发性/难治性滤泡性淋巴瘤(FL)患者的全球性二期研究中,赞德利西布采用间歇给药的方式,以减轻口服PI3Kδ抑制剂每日连续给药时出现的免疫相关不良事件和感染。符合条件的患者如果患有可测量的疾病,且在之前接受过至少两种疗法后病情出现进展,将接受赞德利西布治疗,直至病情恶化或无法耐受。主要疗效终点是客观反应率(ORR),关键的次要疗效终点是反应持续时间(DOR)。我们报告了121例FL患者的治疗情况,这些患者在经过8周的肿瘤剥脱每日给药后,采用间歇给药的方式服用赞德利西布。既往治疗次数的中位数为 3 次(范围为 2-8 次),45% 的患者患有难治性疾病。ORR为73%(95%置信区间[CI],63.9-80.4),完全应答(CR)率为38%(95% CI,29.3-47.3),中位DOR为16.4个月(95% CI,9.5-未达到)。中位随访时间为 14.3 个月(1-30.5 个月),中位无进展生存期为 11.6 个月(95% CI,8.3-未达到)。21名患者(17%)因不良事件中断治疗。3-4级相关毒性包括:6%腹泻、5%肺部感染、3%结肠炎(活检或影像学确诊)、3%皮疹、2%AST升高和1%非感染性肺炎。Zandelisib在重度预处理的复发/难治FL患者中取得了较高的持久应答率。间歇性给药导致严重类相关毒性的发生率相对较低,这为评估Zandelisib作为单药或与不活跃B细胞恶性肿瘤联合用药提供了支持。
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引用次数: 0
Improving CORM technology for the treatment of delayed hemolytic transfusion reaction 改进用于治疗延迟性溶血性输血反应的 CORM 技术。
IF 7.6 2区 医学 Q1 HEMATOLOGY Pub Date : 2024-08-05 DOI: 10.1002/hem3.140
Michela Asperti, Francesca Vinchi
<p>Delayed hemolytic transfusion reaction (DHTR) is a severe and potentially fatal complication triggered by red blood cells (RBC) transfusions<span><sup>1</sup></span> in patients with sickle cell disease (SCD). Transfusions remain a major therapeutic intervention in the clinical management of anemia as well as both acute and chronic disease-related complications in SCD.<span><sup>1-3</sup></span> Typically, DHTR occurs days to weeks after a RBC transfusion due to the sudden destruction of both transfused and patients' RBCs, with a consequent drastic drop in hemoglobin (Hb), seriously threatening the life of SCD patients.<span><sup>4, 5</sup></span> During DHTR with hyperhemolysis, the release of free Hb and heme has deleterious impact on the vasculature, causing vasculo-toxicity and leading to vasculopathy due to intravascular oxidative stress, endothelial damage, increased expression of proadhesive, proinflammatory and chemotactic factors and reduced nitric oxide (NO) bioavailability. Upon RBC exposure, one or more alloantibodies are produced in SCD patients, which contribute to DHTR. In one-third of RBC transfused patients, complement activation—rather than alloantibodies production—plays a role in DHTR, both through the canonic pathway, whereby complement fixed antibody binds to RBCs, and the alternative pathway, whereby free heme-induced TLR4 signaling on endothelial cells activates the complement system.<span><sup>1</sup></span> Patients experience symptoms such as fever, pain, fatigue, mild jaundice or dark urine and a drastic Hb drop. The current treatment options for DHTRs are based on supportive care, erythropoiesis optimization, immunomodulatory treatments, including complement inhibition, steroids, intravenous immunoglobulin, and/or B cell depletion, and future transfusion avoidance, even if the latest may be not always feasible in some clinical conditions related to cardiac or respiratory failure.<span><sup>1, 3</sup></span></p><p>Among the therapeutic strategies proposed to overcome DHTR, carbon monoxide administration in the form of inhalation or carbon-monoxide-releasing molecules (CO-RMs) has shown promising results in preclinical studies.<span><sup>6</sup></span> A plethora of CORMs has been generated, structurally designed with a central transition metal such as iron, manganese, or cobalt, surrounded by CO as a ligand.<span><sup>6</sup></span> CO is a stable molecule that is continuously produced after the catabolism of heme by heme-oxygenases (HO), a family of enzymes with established anti-inflammatory and cytoprotective functions. Mechanistically, CO decreases the expression of proinflammatory and increases the expression of anti-inflammatory cytokines by activating the MKK3/p38β MAPK pathway and inducing PPARγ. In addition, it reduces TLR4 activation by inhibiting TLR4 trafficking, and its interaction with caveolin-1 at the plasma membrane. CO also serves as a bioactive signaling molecule acting as intracellular mediator in
延迟溶血性输血反应(DHTR)是镰状细胞病(SCD)患者因输注红细胞1 而引发的一种严重且可能致命的并发症。1-3 通常情况下,DHTR 在输注 RBC 后数天至数周内发生,原因是输注的 RBC 和患者的 RBC 突然遭到破坏,血红蛋白(Hb)随之急剧下降,严重威胁 SCD 患者的生命、5 在伴有高溶血的 DHTR 期间,游离 Hb 和血红素的释放会对血管产生有害影响,引起血管毒性,并由于血管内氧化应激、内皮损伤、促粘附因子、促炎因子和趋化因子的表达增加以及一氧化氮(NO)生物利用度降低而导致血管病变。SCD 患者在接触红细胞后会产生一种或多种异体抗体,从而导致 DHTR。在三分之一输注了 RBC 的患者中,补体激活(而非同种抗体的产生)在 DHTR 中起着一定的作用,这种作用可通过补体固定抗体与 RBC 结合的 "补体途径 "和内皮细胞上游离血红素诱导的 TLR4 信号激活补体系统的 "替代途径 "1 来实现。目前治疗 DHTR 的方法包括支持性护理、优化红细胞生成、免疫调节治疗(包括补体抑制、类固醇、静脉注射免疫球蛋白和/或 B 细胞耗竭)以及避免输血,尽管在某些与心脏或呼吸衰竭相关的临床病例中,最新的治疗方法并不总是可行、3 在为克服 DHTR 而提出的治疗策略中,一氧化碳吸入或一氧化碳释放分子(CO-RMs)给药已在临床前研究中显示出良好的效果。6 一氧化碳是一种稳定的分子,在血红素氧化酶(HO)分解血红素后持续产生,血红素氧化酶家族具有公认的抗炎和细胞保护功能。从机理上讲,CO 可通过激活 MKK3/p38β MAPK 通路和诱导 PPARγ 减少促炎细胞因子的表达,增加抗炎细胞因子的表达。此外,它还能抑制 TLR4 的贩运及其与质膜上的洞穴素-1 的相互作用,从而减少 TLR4 的激活。CO 还是一种生物活性信号分子,在多种生理功能中充当细胞内介质,包括通过激活可溶性鸟苷酸环化酶扩张血管和保护心脏、通过激活钾通道调节神经系统、控制神经递质以及胃肠道和呼吸道。体外和临床前研究证明,一氧化碳对血管具有显著的抗炎、抗氧化和抗细胞凋亡作用以及血管扩张和抗粘连作用,从而保护血管流动,对 SCD 具有重要意义。在此背景下,Nguyen Kim-Anh 和合作者最近报告了 CORM-401 对 SCD 急性高溶血引起的内皮活化、组织损伤和炎症的有益影响。9 CORM-401 能够携带并向生物系统输送可控量的 CO,从而激活内皮细胞钙信号传导并增加 NO 的生物利用度,从而产生治疗效果、10-12本研究的新颖之处在于建立了体外和体内模型,以反映 SCD 高溶血早期发生的内皮损伤和器官功能障碍。作者利用一种新的体外流体模型,将脐静脉内皮细胞(HUVEC)暴露于含有 RBC 膜衍生颗粒的溶血液中,其中的游离氧化血红蛋白或血红素含量可忽略不计,从而再现了 DHTR 早期的血管活化和功能障碍。将HUVEC细胞预先暴露于CORM-401可显著增加COHb的含量,并防止溶血物诱导的促炎细胞因子(如IL6、IL1和IL8)和粘附分子(包括血管和细胞间细胞粘附分子-1(VCAM-1和ICAM-1))的上调。此外,由于全面降低了氧化应激,它还抑制了急性期氧化还原敏感转录因子核因子红细胞-2相关因子2(Nrf2)的表达。
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引用次数: 0
Bone marrow niches for hematopoietic stem cells 造血干细胞的骨髓龛位。
IF 7.6 2区 医学 Q1 HEMATOLOGY Pub Date : 2024-07-31 DOI: 10.1002/hem3.133
Ana Luísa Pereira, Serena Galli, César Nombela-Arrieta

Hematopoietic stem cells (HSCs) are the cornerstone of the hematopoietic system. HSCs sustain the continuous generation of mature blood derivatives while self-renewing to preserve a relatively constant pool of progenitors throughout life. Yet, long-term maintenance of functional HSCs exclusively takes place in association with their native tissue microenvironment of the bone marrow (BM). HSCs have been long proposed to reside in fixed and identifiable anatomical units found in the complex BM tissue landscape, which control their identity and fate in a deterministic manner. In the last decades, tremendous progress has been made in the dissection of the cellular and molecular fabric of the BM, the structural organization governing tissue function, and the plethora of interactions established by HSCs. Nonetheless, a holistic model of the mechanisms controlling HSC regulation in their niche is lacking to date. Here, we provide an overview of our current understanding of BM anatomy, HSC localization, and crosstalk within local cellular neighborhoods in murine and human tissues, and highlight fundamental open questions on how HSCs functionally integrate in the BM microenvironment.

造血干细胞是造血系统的基石。造血干细胞可持续生成成熟的血液衍生物,同时进行自我更新,在整个生命过程中保持相对恒定的祖细胞池。然而,功能性造血干细胞的长期维持只能与其骨髓(BM)的原生组织微环境相关联。长期以来,人们一直认为造血干细胞居住在复杂的骨髓组织景观中固定且可识别的解剖单元中,这些单元以确定性的方式控制着造血干细胞的身份和命运。在过去几十年中,人们在剖析 BM 的细胞和分子结构、支配组织功能的结构组织以及造血干细胞建立的大量相互作用方面取得了巨大进展。然而,迄今为止还缺乏一个关于造血干细胞在其生态位中调控机制的整体模型。在此,我们将概述目前我们对基底膜解剖、造血干细胞定位以及小鼠和人体组织中局部细胞邻域内相互协作的理解,并强调造血干细胞如何在基底膜微环境中发挥整合功能的基本开放性问题。
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引用次数: 0
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HemaSphere
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