Targeting CD19-positive lymphomas with the antibody-drug conjugate loncastuximab tesirine: preclinical evidence as single agent and in combination therapy.

Antibody-drug conjugates (ADCs) represent one of the most successful therapeutic approaches introduced in clinical practice in the last few years. Loncastuximab tesirine (ADCT-402) is a CD19 targeting ADC, in which the antibody is conjugated through a protease cleavable dipeptide linker to a pyrrolobenzodiazepine (PBD) dimer warhead (SG3199). Based on the results of a phase 2 study, loncastuximab tesirine was recently approved for adult patients with relapsed/refractory large B-cell lymphoma. We assessed the activity of loncastuximab tesirine using in vitro and in vivo models of lymphomas, correlated its activity with CD19 expression levels, and identified combination partners providing synergy with loncastuximab tesirine. Loncastuximab tesirine was tested across 60 lymphoma cell lines. Loncastuximab tesirine had strong cytotoxic activity in B-cell lymphoma cell lines. The in vitro activity was correlated with CD19 expression level and intrinsic sensitivity of cell lines to the ADC's warhead. Loncastuximab tesirine was more potent than other anti-CD19 ADCs (coltuximab ravtansine, huB4-DGN462), albeit the pattern of activity across cell lines was correlated. Loncastuximab tesirine activity was also largely correlated with cell line sensitivity to R-CHOP. Combinatorial in vitro and in vivo experiments identified the benefit of adding loncastuximab tesirine to other agents, especially BCL2 and PI3K inhibitors. Our data support the further development of loncastuximab tesirine as a single agent and in combination for patients affected by mature B-cell neoplasms. The results also highlight the importance of CD19 expression and the existence of lymphoma populations characterized by resistance to multiple therapies.

Chemical screening identifies novel small molecule activators of natural killer cell cytotoxicity against cancer cells.

Natural killer (NK) cells are cytotoxic lymphocytes that play a major role in the innate immune system. NK cells exhibit potent cytotoxic activity against cancer cells and virally infected cells without antigen priming. These unique cytotoxic properties make NK cells a promising therapeutic against cancer. Limitations of NK cell therapy include deficiencies in high clinical efficacy often due to a need for a high NK cell to target cell ratio to achieve effective killing. In order to address the suboptimal efficacy of current adoptive NK cell therapy, a high throughput screen (HTS) was designed and performed to identify drug-like compounds that increase NK cytotoxic activity against tumor cells without affecting the normal cells. This screen was performed in a 384-well plate format utilizing an expanded primary NK cell product and ovarian cancer cells as a target cell (TC) line. Of the 8000 diverse small molecules screened, 16 hits were identified (0.2% hit rate) based on both a robust Z (RZ) score < -3 and a greater than 10% increase in NK cell killing. A validation screen had a confirmation rate of 70%. Select compounds were further validated and characterized by additional cytotoxicity assays including activity against multiple blood cancer and solid tumor cell lines, with no effect on primary human T cells. This work demonstrates that high-throughput screening can be reliably used to identify compounds that increase NK tumoricidal activity in vitro that can be further investigated and translated for potential clinical application. Précis: Our work led to the identification of promising compound that potently increases NK cell-mediated killing of a variety of different cancer cells, but no impact on the killing of normal cells. This compound demonstrates the utility of this assay.

Animation Supported Consent Before Elective Laparoscopic Cholecystectomy.

BACKGROUND: Patient understanding of surgical procedures is often incomplete at the time they are performed, invalidating consent, and exposing healthcare providers to complaints and claims of failure to inform. Remote consultations, language barriers and patient factors can hinder an effective consent pathway. New approaches are needed to support communication and shared decision-making. METHODS: Multi-language digital animations explaining laparoscopic cholecystectomy were introduced at The Royal London Hospital for patients who attended for elective surgery ( www.explainmyprocedure.com/lapchole ). Patients completed questionnaires on the day of their procedure both before and after introduction of the animations. We assessed patient-reported understanding of the procedure, its intended benefits, the possible risks, and alternatives to treatment in 72 consecutive patients, 37 before (no animation group) and after 35 after introducing the animations into the consent pathway (animation group). Patient understanding in the two groups was compared. RESULTS: The two groups were well matched in respect of age, sex and whether English was their first spoken language. The proportions of patients who reported they completely understood the procedure, its benefits, risks, and alternatives in the no animation group were 54, 57, 38 and 24% and in the animation group, 91, 91, 74 and 77%, respectively; p < 0.01 for each comparison. CONCLUSION: The integration of multi-language laparoscopic cholecystectomy video animations into the patient consent pathway was associated with substantial improvement in reported understanding of the procedure, benefits, risks, and alternatives to treatment. This approach can be applied across all surgical disciplines in a standardised manner in an era of accelerated elective work and remote consultations.

Inactivation of the enzyme GSK3α by the kinase IKKi promotes AKT-mTOR signaling pathway that mediates interleukin-1-induced Th17 cell maintenance.

Interleukin-1 (IL-1)-induced activation of the mTOR kinase pathway has major influences on Th17 cell survival, proliferation, and effector function. Via biochemical and genetic approaches, the kinases IKKi and GSK3α were identified as the critical intermediate signaling components for IL-1-induced AKT activation, which in turn activated mTOR. Although insulin-induced AKT activation is known to phosphorylate and inactivate GSK3α and GSK3ß, we found that GSK3α but not GSK3ß formed a constitutive complex to phosphorylate and suppress AKT activation, showing that a reverse action from GSK to AKT can take place. Upon IL-1 stimulation, IKKi was activated to mediate GSK3α phosphorylation at S21, thereby inactivating GSK3α to promote IL-1-induced AKT-mTOR activation. Thus, IKKi has a critical role in Th17 cell maintenance and/or proliferation through the GSK-AKT-mTOR pathway, implicating the potential of IKKi as a therapeutic target.

Expectations of and for Clerkship Directors 2.0: A Collaborative Statement from the Alliance for Clinical Education.

This article presents an update of the collaborative statement on clerkship directors (CDs), first published in 2003, from the national undergraduate medical education organizations that comprise the Alliance for Clinical Education (ACE). The clerkship director remains an essential leader in the education of medical students on core clinical rotations, and the role of the CD has and continues to evolve. The selection of a CD should be an explicit contract between the CD, their department, and the medical school, with each party fulfilling their obligations to ensure the success of the students, the clerkship and of the CD. Educational innovations and accreditation requirements have evolved in the last two decades and therefore this article updates the 2003 standards for what is expected of a CD and provides guidelines for the resources and support to be provided.In their roles as CDs, medical student educators engage in several critical activities: administration, education/teaching, coaching, advising, and mentoring, faculty development, compliance with accreditation standards, and scholarly activity. This article describes (a) the work products that are the primary responsibility of the CD; (b) the qualifications for the CD; (c) the support structure, resources, and personnel that are necessary for the CD to accomplish their responsibilities; (d) incentives and career development for the CD; and (e) the dedicated time that should be provided for the clerkship and the CD to succeed. Given all that should rightfully be expected of a CD, a minimum of 50% of a full-time equivalent is recognized as appropriate. The complexity and needs of the clerkship now require that at least one full-time clerkship administrator (CA) be a part of the CD's team.To better reflect the current circumstances, ACE has updated its recommendations for institutions and departments to have clear standards for what is expected of the director of a clinical clerkship and have correspondingly clear guidelines as to what should be expected for CDs in the support they are provided. This work has been endorsed by each of the eight ACE member organizations.

Research and Scholarly Projects.

The idea of doing a research or scholarly project can be very daunting, however, the satisfaction of seeing a project to its completion is very rewarding. In this article, we provide medical students with guidance on whether they should take on a research or scholarly project during medical school, and how to get started, publish, and then present their project. We also highlight how such a project can benefit an applicant applying for residency training.

Medical Student Leadership in Emergency Medicine.

Emergency medicine is a profession that requires good leadership skills. Emergency physicians must be able to instill confidence in both the staff and patients, inspire the best in others, have the enthusiasm to take on a surplus of responsibilities, and maintain calmness during unexpected circumstances. Accordingly, residency program directors look carefully for leadership qualities and potential among their applicants. Although some people do have a predisposition to lead, leadership can be both learned and taught. In this article, we provide medical students with the tools that will help them acquire those qualities and thus make them more desirable by program directors.

Low-Dose Azacitidine with DNMT1 Level Monitoring to Treat Post-Transplantation Acute Myelogenous Leukemia or Myelodysplastic Syndrome Relapse.

Patients with early relapse of acute myelogenous leukemia (AML) or myelodysplastic syndrome (MDS) after hematopoietic cell transplantation (HCT) have a poor prognosis, and no standard treatment. Twenty-nine patients with early disease recurrence post-transplantation were treated with azacitidine (AZA; median dose, 40 mg/m2/day for 5 to 7 days). At a median follow-up of 6.3 months (range, 1.3 to 41.1 months), 7 patients (27%) had a response to AZA, defined as complete remission, hematologic improvement, or improved donor chimerism. Response occurred after a median of 3 cycles, and the median duration of response was 70 days (range, 26 to 464 days). Median survival was 6.8 months (95% confidence interval, 3.8 to 11.1 months). Survival was similar in the patients receiving an AZA dose ≤40 mg/m2 and those receiving an AZA dose >40 mg/m2. Six patients receiving donor lymphocyte infusion with AZA had a response or stable disease without worsening graft-versus-host-disease. We retrospectively used a flow cytometry assay to explore DNA-methyltransferase-1 in blood mononuclear cells as a potential pharmacodynamic marker to assess intracellular drug targeting in 8 patients. No correlation with AZA dose or response was observed. Low-dose AZA appears to have comparable efficacy to higher-dose AZA post-HCT. A significant proportion of this poor-risk population responded to low-dose AZA, suggesting a dose-independent, noncytotoxic mechanism for antileukemic activity.

Child-Parent Familial Hypercholesterolemia Screening in Primary Care.

BACKGROUND: Child-parent screening for familial hypercholesterolemia has been proposed to identify persons at high risk for inherited premature cardiovascular disease. We assessed the efficacy and feasibility of such screening in primary care practice. METHODS: We obtained capillary blood samples to measure cholesterol levels and to test for familial hypercholesterolemia mutations in 10,095 children 1 to 2 years of age during routine immunization visits. Children were considered to have positive screening results for familial hypercholesterolemia if their cholesterol level was elevated and they had either a familial hypercholesterolemia mutation or a repeat elevated cholesterol level 3 months later. A parent of each child with a positive screening result for familial hypercholesterolemia was considered to have a positive screening result for familial hypercholesterolemia if he or she had the same mutation as the child or, if no mutations were identified, had the higher cholesterol level of the two parents. RESULTS: The use of a prespecified cholesterol cutoff value of 1.53 multiples of the median (MoM, corresponding to a percentile of 99.2) identified 28 children who had positive screening results for familial hypercholesterolemia (0.3% of the 10,095 children; 95% confidence interval [CI], 0.2 to 0.4), including 20 with a familial hypercholesterolemia mutation and 8 with a repeat cholesterol level of at least 1.53 MoM. A total of 17 children who had a cholesterol level of less than 1.53 MoM also had a familial hypercholesterolemia mutation. The overall mutation prevalence was 1 in 273 children (37 in 10,095; 95% CI, 1 in 198 to 1 in 388). The use of an initial cholesterol cutoff value of 1.35 MoM (95th percentile) plus a mutation, or two cholesterol values of at least 1.50 MoM (99th percentile), identified 40 children who had positive screening results for familial hypercholesterolemia (0.4% of the 10,095 children, including 32 children who had a familial hypercholesterolemia mutation and 8 who did not have the mutation) and 40 parents who had positive screening results for familial hypercholesterolemia. CONCLUSIONS: Child-parent screening was feasible in primary care practices at routine child immunization visits. For every 1000 children screened, 8 persons (4 children and 4 parents) were identified as having positive screening results for familial hypercholesterolemia and were consequently at high risk for cardiovascular disease. (Funded by the Medical Research Council.).

Inactivation of 3-hydroxybutyrate dehydrogenase 2 delays zebrafish erythroid maturation by conferring premature mitophagy.

Mitochondria are the site of iron utilization, wherein imported iron is incorporated into heme or iron-sulfur clusters. Previously, we showed that a cytosolic siderophore, which resembles a bacterial siderophore, facilitates mitochondrial iron import in eukaryotes, including zebrafish. An evolutionarily conserved 3-hydroxy butyrate dehydrogenase, 3-hydroxy butyrate dehydrogenase 2 (Bdh2), catalyzes a rate-limiting step in the biogenesis of the eukaryotic siderophore. We found that inactivation of bdh2 in developing zebrafish embryo results in heme deficiency and delays erythroid maturation. The basis for this erythroid maturation defect is not known. Here we show that bdh2 inactivation results in mitochondrial dysfunction and triggers their degradation by mitophagy. Thus, mitochondria are prematurely lost in bdh2-inactivated erythrocytes. Interestingly, bdh2-inactivated erythroid cells also exhibit genomic alterations as indicated by transcriptome analysis. Reestablishment of bdh2 restores mitochondrial function, prevents premature mitochondrial degradation, promotes erythroid development, and reverses altered gene expression. Thus, mitochondrial communication with the nucleus is critical for erythroid development.

Is Emergency Medicine the Right Choice for Me?

One of the hardest decisions a medical student has to make is the choice of specialty. Many studies have explored what influences the choice of emergency medicine (EM) as a specialty. In this article, we elaborate on the most important incentives, including the diversity in patients' presentations, having a defined and flexible schedule, the plasticity in choosing and changing a practice location, and the acuity of care and trauma experience. Additionally, we tackle some of the challenges that emergency physicians face. For instance, having to follow a different thought process than most other physicians, as well as the patients' quality and expectations. We also address some of the concerns regarding the specialty, specifically burnout, stress, and the fear associated with maintaining a career in EM. Finally, we provide students interested in EM with some resources that can provide them with further guidance to decide whether EM is the right choice for them.

A second-generation 15-PGDH inhibitor promotes bone marrow transplant recovery independently of age, transplant dose and granulocyte colony-stimulating factor support.

Hematopoietic stem cell transplantation following myeloablative chemotherapy is a curative treatment for many hematopoietic malignancies. However, profound granulocytopenia during the interval between transplantation and marrow recovery exposes recipients to risks of fatal infection, a significant source of transplant-associated morbidity and mortality. We have previously described the discovery of a small molecule, SW033291, that potently inhibits the prostaglandin degrading enzyme 15-PGDH, increases bone marrow prostaglandin E2, and accelerates hematopoietic recovery following murine transplant. Here we describe the efficacy of (+)-SW209415, a second-generation 15-PGDH inhibitor, in an expanded range of models relevant to human transplantation. (+)-SW209415 is 10,000-fold more soluble, providing the potential for intravenous delivery, while maintaining potency in inhibiting 15-PGDH, increasing in vivo prostaglandin E2, and accelerating hematopoietic regeneration following transplantation. In additional models, (+)-SW209415: (i) demonstrated synergy with granulocyte colony-stimulating factor, the current standard of care; (ii) maintained efficacy as transplant cell dose was escalated; (iii) maintained efficacy when transplant donors and recipients were aged; and (iv) potentiated homing in xenotransplants using human hematopoietic stem cells. (+)-SW209415 showed no adverse effects, no potentiation of in vivo growth of human myeloma and leukemia xenografts, and, on chronic high-dose administration, no toxicity as assessed by weight, blood counts and serum chemistry. These studies provide independent chemical confirmation of the activity of 15-PGDH inhibitors in potentiating hematopoietic recovery, extend the range of models in which inhibiting 15-PGDH demonstrates activity, allay concerns regarding potential for adverse effects from increasing prostaglandin E2, and thereby, advance 15-PGDH as a therapeutic target for potentiating hematopoietic stem cell transplantation.

Randomized trial of preventive angioplasty in myocardial infarction.

BACKGROUND: In acute ST-segment elevation myocardial infarction (STEMI), the use of percutaneous coronary intervention (PCI) to treat the artery responsible for the infarct (infarct, or culprit, artery) improves prognosis. The value of PCI in noninfarct coronary arteries with major stenoses (preventive PCI) is unknown. METHODS: From 2008 through 2013, at five centers in the United Kingdom, we enrolled 465 patients with acute STEMI (including 3 patients with left bundle-branch block) who were undergoing infarct-artery PCI and randomly assigned them to either preventive PCI (234 patients) or no preventive PCI (231 patients). Subsequent PCI for angina was recommended only for refractory angina with objective evidence of ischemia. The primary outcome was a composite of death from cardiac causes, nonfatal myocardial infarction, or refractory angina. An intention-to-treat analysis was used. RESULTS: By January 2013, the results were considered conclusive by the data and safety monitoring committee, which recommended that the trial be stopped early. During a mean follow-up of 23 months, the primary outcome occurred in 21 patients assigned to preventive PCI and in 53 patients assigned to no preventive PCI (infarct-artery-only PCI), which translated into rates of 9 events per 100 patients and 23 per 100, respectively (hazard ratio in the preventive-PCI group, 0.35; 95% confidence interval [CI], 0.21 to 0.58; P<0.001). Hazard ratios for the three components of the primary outcome were 0.34 (95% CI, 0.11 to 1.08) for death from cardiac causes, 0.32 (95% CI, 0.13 to 0.75) for nonfatal myocardial infarction, and 0.35 (95% CI, 0.18 to 0.69) for refractory angina. CONCLUSIONS: In patients with STEMI and multivessel coronary artery disease undergoing infarct-artery PCI, preventive PCI in noninfarct coronary arteries with major stenoses significantly reduced the risk of adverse cardiovascular events, as compared with PCI limited to the infarct artery. (Funded by Barts and the London Charity; PRAMI Current Controlled Trials number, ISRCTN73028481.).

Implementing a third-year emergency medicine medical student curriculum.

BACKGROUND: Emergency medicine (EM) is commonly introduced in the fourth year of medical school because of a perceived need to have more experienced students in the complex and dynamic environment of the emergency department. However, there is no evidence supporting the optimal time or duration for an EM rotation, and a number of institutions offer third-year rotations. OBJECTIVE: A recently published syllabus provides areas of knowledge, skills, and attitudes that third-year EM rotation directors can use to develop curricula. This article expands on that syllabus by providing a comprehensive curricular guide for the third-year medical student rotation with a focus on implementation. DISCUSSION: Included are consensus-derived learning objectives, discussion of educational methods, considerations for implementation, and information on feedback and evaluation as proposed by the Clerkship Directors in Emergency Medicine Third-Year Curriculum Work Group. External validation results, derived from a survey of third-year rotation directors, are provided in the form of a content validity index for each content area. CONCLUSIONS: This consensus-derived curricular guide can be used by faculty who are developing or revising a third-year EM medical student rotation and provide guidance for implementing this curriculum at their institution.

Humans accumulate microsatellite instability with acquired loss of MLH1 protein in hematopoietic stem and progenitor cells as a function of age.

Hematopoietic stem and progenitor cells (HPCs) are necessary for long-term survival. Genomic instability and persistent DNA damage may cause loss of adult stem cell function. The mismatch repair (MMR) pathway increases replication fidelity and defects have been implicated in malignant hematopoietic diseases. Little, however, is known about the role MMR pathway failure plays in the aging process of human HPCs. We hypothesized that loss of MMR occurs in HPCs as a process of human aging. We examined microsatellite instability and expression of the MMR genes MutL homologue 1 (MLH1) and MutS homologue 2 (MSH2) in HPCs and colony-forming cell-derived clones (CFCs) from human donors aged 0 to 86 years. CFCs from donors > 45 years had a greater frequency of microsatellite instability and CD34(+) progenitors lacking MLH1 expression and protein than individuals ≤ 45 years. Loss of MSH2 did not correlate with age. Thus, a potentially early event in the normal human aging process is microsatellite instability accumulation in normal human HPCs associated with the loss of MLH1 protein expression.

Wnt signaling is involved in 6-benzylthioinosine-induced AML cell differentiation.

BACKGROUND: We previously demonstrated that 6-benzylthioinosine (6-BT) could induce the differentiation of a subset of acute myeloid leukemia (AML) cell lines and primary AML cells regardless of their cytogenetics. In this study we investigated whether Wnt signaling pathways played roles in 6-BT-induced differentiation of AML cells. METHODS: We induced differentiation of HL-60 leukemic cells and primary AML cells in vitro using 6-BT. Real-time PCR (qPCR), western blot, and luciferase assays were used to examine the molecules' expression and biological activity in canonical and noncanonical Wnt signaling pathways. AML cell differentiation was measured by the Nitroblue tetrozolium (NBT) reduction assay. RESULTS: 6-BT regulated the expression of both canonical and non-canonical Wnt signaling molecules in HL-60 cells. Both 6-BT and all-trans-retinoic-acid (ATRA) reduced canonical Wnt signaling and activated noncanonical Wnt/Ca2+ signaling in HL-60 cells. Pre-treatment of HL-60 cells with an inhibitor of glycogen synthase kinase-3ß (GSK-3ß), which activated canonical Wnt signaling, partly abolished the differentiation of HL-60 cells induced by 6-BT. Pre-treatment of HL-60 cells with an inhibitor of protein kinase C (PKC), resulting in inactivation of non-canonical Wnt/Ca2+ signaling, abolished 6-BT-induced differentiation of HL-60 cells. Several molecules in the non-canonical Wnt/Ca2+ pathway were detected in bone marrow samples from AML patients, and the expression of FZD4, FZD5, Wnt5a and RHOU were significantly reduced in newly diagnosed AML samples compared with normal controls. CONCLUSIONS: Both canonical and non-canonical Wnt signaling were involved in 6-BT-induced differentiation of HL-60 cells, and played opposite roles in this process. Wnt signaling could be involved in the pathogenesis of AML not only by regulating self-renewal of hematopoietic stem cells, but also by playing a role in the differentiation of AML cells.

Single cell and bulk RNA expression analyses identify enhanced hexosamine biosynthetic pathway and O-GlcNAcylation in acute myeloid leukemia blasts and stem cells.

Introduction: Acute myeloid leukemia (AML) is the most common acute leukemia in adults with an overall poor prognosis and high relapse rate. Multiple factors including genetic abnormalities, differentiation defects and altered cellular metabolism contribute to AML development and progression. Though the roles of oxidative phosphorylation and glycolysis are defined in AML, the role of the hexosamine biosynthetic pathway (HBP), which regulates the O-GlcNAcylation of cytoplasmic and nuclear proteins, remains poorly defined. Methods: We studied the expression of the key enzymes involved in the HBP in AML blasts and stem cells by RNA sequencing at the single-cell and bulk level. We performed flow cytometry to study OGT protein expression and global O-GlcNAcylation. We studied the functional effects of inhibiting O-GlcNAcylation on transcriptional activation in AML cells by Western blotting and real time PCR and on cell cycle by flow cytometry. Results: We found higher expression levels of the key enzymes in the HBP in AML as compared to healthy donors in whole blood. We observed elevated O-GlcNAc Transferase (OGT) and O-GlcNAcase (OGA) expression in AML stem and bulk cells as compared to normal hematopoietic stem and progenitor cells (HSPCs). We also found that both AML bulk cells and stem cells show significantly enhanced OGT protein expression and global O-GlcNAcylation as compared to normal HSPCs, validating our in silico findings. Gene set analysis showed substantial enrichment of the NF-κB pathway in AML cells expressing high OGT levels. Inhibition of O-GlcNAcylation decreased NF-κB nuclear translocation and the expression of selected NF-κB-dependent genes controlling cell cycle. It also blocked cell cycle progression suggesting a link between enhanced O-GlcNAcylation and NF-κB activation in AML cell survival and proliferation. Discussion: Our study suggests the HBP may prove a potential target, alone or in combination with other therapeutic approaches, to impact both AML blasts and stem cells. Moreover, as insufficient targeting of AML stem cells by traditional chemotherapy is thought to lead to relapse, blocking HBP and O-GlcNAcylation in AML stem cells may represent a novel promising target to control relapse.