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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.^{1, 2} 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.¹ 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.² Both methods approach 80%–90% of editing efficiency and allow to perform downstream in vitro experiments on edited leukemic cells.

Application of a similar RNP-based editing approach to the widely used murine model of CLL, the Eμ-TCL1 transgenic mice,³ represents a valuable and versatile tool to explore CLL biology in vivo. Examples illustrating its feasibility has been first shown in studies by Chakraborthy et al. and Martines et al.^{4, 5} The published method consists in preactivating primary CD19⁺CD5⁺ leukemic B cells by TLR9 agonist CpG ODN-1668, followed by RNP electroporation and intraperitoneal injection of 30 × 10⁶ electroporated cells to promote expansion of edited leukemic cells in vivo. Despite this method has been proven effective, it has not been set up to expand edited TCL1 cells in vitro. This is associated with high experimental costs and does not allow to perform functional analysis of gene editing phenotype prior to the in vivo transfer, which eventually becomes not feasible if edited cells are unfit in vivo.

Hence, we envisioned a new approach that would allow to expand RNP-electroporated TCL1 cells in vitro 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,⁵ does not allow to expand them in vitro (Supporting Information S1: Figure S1). We thus evaluated

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.

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.

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.

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.¹ Among people living with SCD, stigma affects every aspect of daily living and may negatively impact relationships with peers, friends, and family.²

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

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.

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 them¹ 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.

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.

First, a face-to-face fixed training commitment provides a protected space and time and a positive social pressure to engage now. 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.

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.

Third, face-to-face training helps trainees to learn from each other. Interacting and connecting with others is essential for learning individually and collectively.² Different ‘learni

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.¹ 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.²

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.^3-8

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.

We deploy a two-step validation process that has been well described.^{3, 8} 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

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.

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.

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.

Delayed hemolytic transfusion reaction (DHTR) is a severe and potentially fatal complication triggered by red blood cells (RBC) transfusions¹ 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.^1-3 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.^{4, 5} 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.¹ 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.^{1, 3}

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.⁶ 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.⁶ 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

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.