Bitter Taste Receptor Agonist Denatonium Inhibits Stemness Characteristics in Hematopoietic Stem/Progenitor Cells.

Bone marrow microenvironmental stimuli profoundly impact hematopoietic stem cell fate and biology. As G protein-coupled receptors, the bitter taste receptors (TAS2Rs) are key in transmitting extracellular stimuli into an intracellular response, within the oral cavity but also in extraoral tissues. Their expression in the bone marrow (BM)-derived cells suggests their involvement in sensing the BM microenvironmental fluctuation. In the present study, we demonstrated that umbilical cord blood (UCB)-derived CD34+ cells express fully functional TAS2Rs along with the signal transduction cascade components and their activation by the prototypical agonist, denatonium benzoate, significantly modulated genes involved in stemness maintenance and regulation of cell trafficking. The activation of these specific pathways was confirmed in functional in vitro experiments. Denatonium exposure exerted an antiproliferative effect on UCB-derived CD34+ cells, mainly affecting the most undifferentiated progenitor frequency. It also reduced their clonogenicity and repopulating potential in vitro. In addition, the TAS2R signaling activation impaired the UCB-derived CD34+ cell trafficking, mainly reducing the migration toward the chemoattractant agent CXCL12 and modulating the expression of the adhesion molecules CD62L, CD49d, and CD29. In conclusion, our results in UCB-derived CD34+ cells expand the observation of TAS2R expression in the setting of BM-resident cells and shed light on the role of TAS2Rs in the extrinsic regulation of hematopoietic stem cell functions.

Expanding the phenotype of Brunner syndrome from childhood to adulthood: Description of the second pediatric patient and his mother.

Brunner syndrome is a recessive X-linked disorder caused by pathogenic variants in the monoamine oxidase A gene (MAOA). It is characterized by distinctive aggressive behavior, mild intellectual disability, sleep disturbances, and typical biochemical alterations deriving from the impaired monoamine metabolism. We herein describe a 5-year-old boy with developmental delay, autistic features, and myoclonic epilepsy, and his mother, who had mild intellectual disability and recurrent episodes of palpitations, headache, abdominal pain, and abdominal bloating. Whole exome sequencing allowed detection of the maternally-inherited variant c.410A>G, (p.Glu137Gly) in the MAOA gene. The subsequent biochemical studies confirmed the MAOA deficiency both in the child and his mother. Given the serotonergic symptoms associated with high serotonin levels found in the mother, treatment with a serotonin reuptake inhibitor and dietary modifications were carried out, resulting in regression of the biochemical abnormalities and partial reduction of symptoms. Our report expands the phenotypic spectrum of Brunner disease, bringing new perspectives on the behavioral and neurodevelopmental phenotype from childhood to adulthood.

Tracking Response and Resistance in Acute Myeloid Leukemia through Single-Cell DNA Sequencing Helps Uncover New Therapeutic Targets.

Acute myeloid leukemia (AML) is an aggressive hematologic neoplasia with a complex polyclonal architecture. Among driver lesions, those involving the FLT3 gene represent the most frequent mutations identified at diagnosis. The development of tyrosine kinase inhibitors (TKIs) has improved the clinical outcomes of FLT3-mutated patients (Pt). However, overcoming resistance to these drugs remains a challenge. To unravel the molecular mechanisms underlying therapy resistance and clonal selection, we conducted a longitudinal analysis using a single-cell DNA sequencing approach (MissionBioTapestri® platform, San Francisco, CA, USA) in two patients with FLT3-mutated AML. To this end, samples were collected at the time of diagnosis, during TKI therapy, and at relapse or complete remission. For Pt #1, disease resistance was associated with clonal expansion of minor clones, and 2nd line TKI therapy with gilteritinib provided a proliferative advantage to the clones carrying NRAS and KIT mutations, thereby responsible for relapse. In Pt #2, clonal architecture was less complex, and 1st line TKI therapy with midostaurin was able to eradicate the leukemic clones. Our results corroborate previous findings about clonal selection driven by TKIs, highlighting the importance of a deeper characterization of individual clonal architectures for choosing the best treatment plan for personalized approaches aimed at optimizing outcomes.

Targeting Leukemic Stem Cells in Chronic Myeloid Leukemia: Is It Worth the Effort?

Chronic myeloid leukemia (CML) is a classical example of stem cell cancer since it arises in a multipotent hematopoietic stem cell upon the acquisition of the t(9;22) chromosomal translocation, that converts it into a leukemic stem cell (LSC). The resulting BCR-ABL1 fusion gene encodes a deregulated tyrosine kinase that is recognized as the disease driver. Therapy with tyrosine kinase inhibitors (TKIs) eliminates progenitor and more differentiated cells but fails to eradicate quiescent LSCs. Thus, although many patients obtain excellent responses and a proportion of them can even attempt treatment discontinuation (treatment free remission [TFR]) after some years of therapy, LSCs persist, and represent a potentially dangerous reservoir feeding relapse and hampering TFR. Over the past two decades, intensive efforts have been devoted to the characterization of CML LSCs and to the dissection of the cell-intrinsic and -extrinsic mechanisms sustaining their persistence, in an attempt to find druggable targets enabling LSC eradication. Here we provide an overview and an update on these mechanisms, focusing in particular on the most recent acquisitions. Moreover, we provide a critical appraisal of the clinical relevance and feasibility of LSC targeting in CML.

The Role of Hypoxic Bone Marrow Microenvironment in Acute Myeloid Leukemia and Future Therapeutic Opportunities.

Acute myeloid leukemia (AML) is a hematologic malignancy caused by a wide range of alterations responsible for a high grade of heterogeneity among patients. Several studies have demonstrated that the hypoxic bone marrow microenvironment (BMM) plays a crucial role in AML pathogenesis and therapy response. This review article summarizes the current literature regarding the effects of the dynamic crosstalk between leukemic stem cells (LSCs) and hypoxic BMM. The interaction between LSCs and hypoxic BMM regulates fundamental cell fate decisions, including survival, self-renewal, and proliferation capacity as a consequence of genetic, transcriptional, and metabolic adaptation of LSCs mediated by hypoxia-inducible factors (HIFs). HIF-1α and some of their targets have been associated with poor prognosis in AML. It has been demonstrated that the hypoxic BMM creates a protective niche that mediates resistance to therapy. Therefore, we also highlight how hypoxia hallmarks might be targeted in the future to hit the leukemic population to improve AML patient outcomes.

Synthesis of Novel Tryptamine Derivatives and Their Biological Activity as Antitumor Agents.

We synthesized five novel tryptamine derivatives characterized by the presence of an azelayl chain or of a 1,1,1-trichloroethyl group, in turn connected to another heterocyclic scaffold. The combination of tryptamin-, 1,1,1-trichloroethyl- and 2-aminopyrimidinyl- moieties produced compound 9 identified as the most active compound in hematological cancer cell lines (IC50 = 0.57-65.32 µM). Moreover, keeping constant the presence of the tryptaminic scaffold and binding it to the azelayl moiety, the compounds maintain biological activity. Compound 13 is still active against hematological cancer cell lines and shows a selective effect only on HT29 cells (IC50 = 0.006 µM) among solid tumor models. Compound 14 loses activity on all leukemic lines, while showing a high level of toxicity on all solid tumor lines tested (IC50 0.0015-0.469 µM).

Recent Advances in the Molecular Biology of Systemic Mastocytosis: Implications for Diagnosis, Prognosis, and Therapy.

In recent years, molecular characterization and management of patients with systemic mastocytosis (SM) have greatly benefited from the application of advanced technologies. Highly sensitive and accurate assays for KIT D816V mutation detection and quantification have allowed the switch to non-invasive peripheral blood testing for patient screening; allele burden has prognostic implications and may be used to monitor therapeutic efficacy. Progress in genetic profiling of KIT, together with the use of next-generation sequencing panels for the characterization of associated gene mutations, have allowed the stratification of patients into three subgroups differing in terms of pathogenesis and prognosis: i) patients with mast cell-restricted KIT D816V; ii) patients with multilineage KIT D816V-involvement; iii) patients with "multi-mutated disease". Thanks to these findings, new prognostic scoring systems combining clinical and molecular data have been developed. Finally, non-genetic SETD2 histone methyltransferase loss of function has recently been identified in advanced SM. Assessment of SETD2 protein levels and activity might provide prognostic information and has opened new research avenues exploring alternative targeted therapeutic strategies. This review discusses how progress in recent years has rapidly complemented previous knowledge improving the molecular characterization of SM, and how this has the potential to impact on patient diagnosis and management.

Aneuploidy: Cancer strength or vulnerability?

Aneuploidy is a very rare and tissue-specific event in normal conditions, occurring in a low number of brain and liver cells. Its frequency increases in age-related disorders and is one of the hallmarks of cancer. Aneuploidy has been associated with defects in the spindle assembly checkpoint (SAC). However, the relationship between chromosome number alterations, SAC genes and tumor susceptibility remains unclear. Here, we provide a comprehensive review of SAC gene alterations at genomic and transcriptional level across human cancers and discuss the oncogenic and tumor suppressor functions of aneuploidy. SAC genes are rarely mutated but frequently overexpressed, with a negative prognostic impact on different tumor types. Both increased and decreased SAC gene expression show oncogenic potential in mice. SAC gene upregulation may drive aneuploidization and tumorigenesis through mitotic delay, coupled with additional oncogenic functions outside mitosis. The genomic background and environmental conditions influence the fate of aneuploid cells. Aneuploidy reduces cellular fitness. It induces growth and contact inhibition, mitotic and proteotoxic stress, cell senescence and production of reactive oxygen species. However, aneuploidy confers an evolutionary flexibility by favoring genome and chromosome instability (CIN), cellular adaptation, stem cell-like properties and immune escape. These properties represent the driving force of aneuploid cancers, especially under conditions of stress and pharmacological pressure, and are currently under investigation as potential therapeutic targets. Indeed, promising results have been obtained from synthetic lethal combinations exploiting CIN, mitotic defects, and aneuploidy-tolerating mechanisms as cancer vulnerability.

Aneuploid acute myeloid leukemia exhibits a signature of genomic alterations in the cell cycle and protein degradation machinery.

BACKGROUND: Aneuploidy occurs in more than 20% of acute myeloid leukemia (AML) cases and correlates with an adverse prognosis. METHODS: To understand the molecular bases of aneuploid acute myeloid leukemia (A-AML), this study examined the genomic profile in 42 A-AML cases and 35 euploid acute myeloid leukemia (E-AML) cases. RESULTS: A-AML was characterized by increased genomic complexity based on exonic variants (an average of 26 somatic mutations per sample vs 15 for E-AML). The integration of exome, copy number, and gene expression data revealed alterations in genes involved in DNA repair (eg, SLX4IP, RINT1, HINT1, and ATR) and the cell cycle (eg, MCM2, MCM4, MCM5, MCM7, MCM8, MCM10, UBE2C, USP37, CK2, CK3, CK4, BUB1B, NUSAP1, and E2F) in A-AML, which was associated with a 3-gene signature defined by PLK1 and CDC20 upregulation and RAD50 downregulation and with structural or functional silencing of the p53 transcriptional program. Moreover, A-AML was enriched for alterations in the protein ubiquitination and degradation pathway (eg, increased levels of UHRF1 and UBE2C and decreased UBA3 expression), response to reactive oxygen species, energy metabolism, and biosynthetic processes, which may help in facing the unbalanced protein load. E-AML was associated with BCOR/BCORL1 mutations and HOX gene overexpression. CONCLUSIONS: These findings indicate that aneuploidy-related and leukemia-specific alterations cooperate to tolerate an abnormal chromosome number in AML, and they point to the mitotic and protein degradation machineries as potential therapeutic targets.

A quantitative transcriptome reference map of the normal human brain.

We performed an innovative systematic meta-analysis of 60 gene expression profiles of whole normal human brain, to provide a quantitative transcriptome reference map of it, i.e. a reference typical value of expression for each of the 39,250 known, mapped and 26,026 uncharacterized (unmapped) transcripts. To this aim, we used the software named Transcriptome Mapper (TRAM), which is able to generate transcriptome maps based on gene expression data from multiple sources. We also analyzed differential expression by comparing the brain transcriptome with those derived from human foetal brain gene expression, from a pool of human tissues (except the brain) and from the two normal human brain regions cerebellum and cerebral cortex, which are two of the main regions severely affected when cognitive impairment occurs, as happens in the case of trisomy 21. Data were downloaded from microarray databases, processed and analyzed using TRAM software and validated in vitro by assaying gene expression through several magnitude orders by 'real-time' reverse transcription polymerase chain reaction (RT-PCR). The excellent agreement between in silico and experimental data suggested that our transcriptome maps may be a useful quantitative reference benchmark for gene expression studies related to the human brain. Furthermore, our analysis yielded biological insights about those genes which have an intrinsic over-/under-expression in the brain, in addition offering a basis for the regional analysis of gene expression. This could be useful for the study of chromosomal alterations associated to cognitive impairment, such as trisomy 21, the most common genetic cause of intellectual disability.

Communicating Adolescent and Young Adult Oncology Treatment Guidelines in Practice: The Importance of Documentation for Ensuring Positive Impacts on Oncology Care.

Purpose: Improvements in outcomes for adolescent and young adult (AYA) oncology patients have lagged behind those of other age-specific cancer populations. Research has indicated that low availability of clinical trials, biological differences of this age-group, and several psychosocial factors including higher emotional distress impact outcomes. To improve care and survival rates for these patients, hospitals have implemented AYA oncology programs. The current study evaluated documentation of care in an AYA program housed in an academic medical center based on three areas emphasized in the National Comprehensive Cancer Network's Clinical Practice Guidelines in Oncology for AYAs: clinical trial enrollment, fertility, and psychosocial care. Methods: Retrospective chart reviews were conducted for 45 patients treated before the start of the AYA oncology program and 45 patients treated after program initiation. Patients aged 15-39 years with a diagnosis of a malignant tumor were included. Variables evaluated included documentation of clinical trial enrollment, fertility preservation and sexual health considerations, and behavioral health referrals. Results: Documentation of most clinical trial and fertility variables did not significantly improve from pre- to post-program, although a higher number of patients had these variables documented post-program. Behavioral health referrals increased significantly from 52.8% pre-program to 95.4% post-program. Conclusion: Access to behavioral health care improved the most following implementation of our AYA program, which is likely because of the integration of a dedicated psychologist for AYAs when the program began. The practice of guideline-based care for this population can be better assessed and improved with designated behavioral health providers and more systematic documentation processes.

SETD2 non genomic loss of function in advanced systemic mastocytosis is mediated by an Aurora kinase A/MDM2 axis and can be therapeutically targeted.

BACKGROUND: The SETD2 tumor suppressor gene encodes a histone methyltransferase that safeguards transcription fidelity and genomic integrity via trimethylation of histone H3 lysine 36 (H3K36Me3). SETD2 loss of function has been observed in solid and hematologic malignancies. We have recently reported that most patients with advanced systemic mastocytosis (AdvSM) and some with indolent or smoldering SM display H3K36Me3 deficiency as a result of a reversible loss of SETD2 due to reduced protein stability. METHODS: Experiments were conducted in SETD2-proficient (ROSAKIT D816V) and -deficient (HMC-1.2) cell lines and in primary cells from patients with various SM subtypes. A short interfering RNA approach was used to silence SETD2 (in ROSAKIT D816V cells), MDM2 and AURKA (in HMC-1.2 cells). Protein expression and post-translational modifications were assessed by WB and immunoblotting. Protein interactions were tested by using co-immunoprecipitation. Apoptotic cell death was evaluated by flow cytometry after annexin V and propidium iodide staining, respectively. Drug cytotoxicity in in vitro experiments was evaluated by clonogenic assays. RESULTS: Here, we show that the proteasome inhibitors suppress cell growth and induce apoptosis in neoplastic mast cells by promoting SETD2/H3K36Me3 re-expression. Moreover, we found that Aurora kinase A and MDM2 are implicated in SETD2 loss of function in AdvSM. In line with this observation, direct or indirect targeting of Aurora kinase A with alisertib or volasertib induced reduction of clonogenic potential and apoptosis in human mast cell lines and primary neoplastic cells from patients with AdvSM. Efficacy of Aurora A or proteasome inhibitors was comparable to that of the KIT inhibitor avapritinib. Moreover, combination of alisertib (Aurora A inhibitor) or bortezomib (proteasome inhibitor) with avapritinib allowed to use lower doses of each drug to achieve comparable cytotoxic effects. CONCLUSIONS: Our mechanistic insights into SETD2 non-genomic loss of function in AdvSM highlight the potential value of novel therapeutic targets and agents for the treatment of patients who fail or do not tolerate midostaurin or avapritinib.

Transport of functionally appropriate tools by capuchin monkeys (Cebus apella).

Capuchin monkeys (Cebus sp.) are notable among New World monkeys for their widespread use of tools. Like chimpanzees, they use both hammer tools and insertion tools in the wild to acquire food that would be unobtainable otherwise. Recent evidence indicates that capuchins transport stones to anvil sites and use the most functionally efficient stones to crack nuts. We further investigated capuchins' assessment of functionality by testing their ability to select a tool that was appropriate for two different tool-use tasks: A stone for a hammer task and a stick for an insertion task. To select the appropriate tools, the monkeys investigated a baited tool-use apparatus (insertion or hammer), traveled to a location in their enclosure where they could no longer see the apparatus, made a selection between two tools (stick or stone), and then could transport the tool back to the apparatus to obtain a walnut. We incorporated tool transport and the lack of a visual cue into the design to assess willingness to transport the tools and the monkeys' memory for the proper tool. Six brown capuchins (Cebus apella) were first trained to select and use the appropriate tool for each apparatus. Four animals completed training and were then tested by allowing them to view a baited apparatus and then travel to a location 8 m distant where they could select a tool while out of view of the apparatus. All four monkeys chose the correct tool significantly more than expected and transported the tools back to the apparatus. Results confirm capuchins' propensity for transporting tools, demonstrate their capacity to select the functionally appropriate tool for two different tool-use tasks, and indicate that they can retain the memory of the correct choice during a travel time of several seconds.

Overcoming Resistance to Kinase Inhibitors: The Paradigm of Chronic Myeloid Leukemia.

Protein kinases (PKs) play crucial roles in cellular proliferation and survival, hence their deregulation is a common event in the pathogenesis of solid and hematologic malignancies. Targeting PKs has been a promising strategy in cancer treatment, and there are now a variety of approved anticancer drugs targeting PKs. However, the phenomenon of resistance remains an obstacle to be addressed and overcoming resistance is a goal to be achieved. Chronic myeloid leukemia (CML) is the first as well as one of the best examples of a cancer that can be targeted by molecular therapy; hence, it can be used as a model disease for other cancers. This review aims to summarize up-to-date knowledge on the main mechanisms implicated in resistance to PK inhibitory therapies and to outline the main strategies that are being explored to overcome resistance. The importance of molecular diagnostics and disease monitoring in counteracting resistance will also be discussed.

CDC20 in and out of mitosis: a prognostic factor and therapeutic target in hematological malignancies.

Cell division cycle 20 homologue (CDC20) is a well-known regulator of cell cycle, as it controls the correct segregation of chromosomes during mitosis. Many studies have focused on the biological role of CDC20 in cancer development, as alterations of its functionality have been linked to genomic instability and evidence demonstrated that high CDC20 expression levels are associated with poor overall survival in solid cancers. More recently, novel CDC20 functions have been demonstrated or suggested, including the regulation of apoptosis and stemness properties and a correlation with immune cell infiltration. Here, we here summarize and discuss the role of CDC20 inside and outside mitosis, starting from its network of interacting proteins. In the last years, CDC20 has also attracted more interest in the blood cancer field, being overexpressed and showing an association with prognosis both in myeloid and lymphoid malignancies. Preclinical findings showed that selective CDC20 and APC/CCDC20/APC/CCDH1 inhibitors, namely Apcin and proTAME, are effective against lymphoma and multiple myeloma cells, resulting in mitotic arrest and apoptosis and synergizing with clinically-relevant drugs. The evidence and hypothesis presented in this review provide the input for further biological and chemical studies aiming to dissect novel potential CDC20 roles and targeting strategies in hematological malignancies.

Combined Inhibition of Polo-Like Kinase-1 and Wee1 as a New Therapeutic Strategy to Induce Apoptotic Cell Death in Neoplastic Mast Cells.

Systemic mastocytosis (SM) is due to the pathologic accumulation of neoplastic mast cells in one or more extracutaneous organ(s). Although midostaurin, a multikinase inhibitor active against both wild-type and D816V-mutated KIT, improves organ damage and symptoms, a proportion of patients relapse or have resistant disease. It is well known that Aurora kinase A (AKA) over-expression promotes tumorigenesis, but its role in the pathogenesis of systemic mastocytosis (SM) has not yet been investigated. Evidence from the literature suggests that AKA may confer cancer cell chemo-resistance, inhibit p53, and enhance Polo-like kinase 1 (Plk1), CDK1, and cyclin B1 to promote cell cycle progression. In this study, we aimed to investigate the pathogenetic role of AKA and Plk1 in the advanced forms of SM. We demonstrate here, for the first time, that SM cell lines display hyper-phosphorylated AKA and Plk1. Danusertib (Aurora kinase inhibitor) and volasertib (Plk1 inhibitor) inhibited growth and induced apoptotic cell death in HMC-1.1 and -1.2 cells. Their growth-inhibitory effects were associated with cell cycle arrest and the activation of apoptosis. Cell cycle arrest was associated with increased levels of phospho-Wee1. Wee1 inhibition by MK1775 after 24 h treatment with danusertib or volasertib, when cells were arrested in G2 phase and Wee1, was overexpressed and hyper-activated, resulting in a significantly higher rate of apoptosis than that obtained from concomitant treatment with danusertib or volasertib + MK1775 for 48 h. In conclusion, Plk1 and AKA, alone or together with Wee1, are attractive therapeutic targets in neoplastic MCs. Repurposing Plk1 or AKA ± Wee1 inhibitors in advanced clinical development for other indications is a therapeutic strategy worthy of being explored, in order to improve the outcome of patients with advanced SM.

Polo-like kinase-1, Aurora kinase A and WEE1 kinase are promising druggable targets in CML cells displaying BCR::ABL1-independent resistance to tyrosine kinase inhibitors.

In chronic myeloid leukemia (CML), Aurora kinase A and Polo like kinase 1 (PLK1), two serine-threonine kinases involved in the maintenance of genomic stability by preserving a functional G2/M checkpoint, have been implicated in BCR::ABL1-independent resistance to the tyrosine kinase inhibitor (TKI) imatinib mesylate and in leukemic stem cell (LSC) persistence. It can be speculated that the observed deregulated activity of Aurora A and Plk1 enhances DNA damage, promoting the occurrence of additional genomic alterations contributing to TKI resistance and ultimately driving progression from chronic phase to blast crisis (BC). In this study, we propose a new therapeutic strategy based on the combination of Aurora kinase A or PLK1 inhibition with danusertib or volasertib, respectively, and WEE1 inhibition with AZD1775. Danusertib and volasertib used as single drugs induced apoptosis and G2/M-phase arrest, associated with accumulation of phospho-WEE1. Subsequent addition of the WEE1 inhibitor AZD1775 in combination significantly enhanced the induction of apoptotic cell death in TKI-sensitive and -resistant cell lines as compared to both danusertib and volasertib alone and to the simultaneous combination. This schedule indeed induced a significant increase of the DNA double-strand break marker γH2AX, forcing the cells through successive replication cycles ultimately resulting in apoptosis. Finally, combination of danusertib or volasertib+AZD1775 significantly reduced the clonogenic potential of CD34+ CML progenitors from BC patients. Our results may have implications for the development of innovative therapeutic approaches aimed to improve the outcomes of patients with multi-TKI-resistant or BC CML.

Droplet digital PCR for the detection of second-generation tyrosine kinase inhibitor-resistant BCR::ABL1 kinase domain mutations in chronic myeloid leukemia.

One of the indications for BCR::ABL1 mutation testing in chronic myeloid leukemia (CML) is when tyrosine kinase inhibitor therapy (TKI) needs to be changed for unsatisfactory response. In this study, we evaluated a droplet digital PCR (ddPCR)-based multiplex strategy for the detection and quantitation of transcripts harbouring mutations conferring resistance to second-generation TKIs (2GTKIs). Parallel quantitation of e13a2, e14a2 and e1a2 BCR::ABL1 fusion transcripts enables to express results as percentage of mutation positive- over total BCR::ABL1 transcripts. We determined the limit of blank in 60 mutation-negative samples. Accuracy was demonstrated by further analysis of 48 samples already studied by next generation sequencing (NGS). Mutations could be called down to 0.5% and across 3-logs of BCR::ABL1 levels. Retrospective review of BCR::ABL1 NGS results in 513 consecutive CML patients with non-optimal response to first- or second-line TKI therapy suggested that a ddPCR-based approach targeted against 2GTKI-resistant mutations would score samples as mutation-negative in 22% of patients with warning response to imatinib but only in 6% of patients with warning response to 2GTKIs. We conclude ddPCR represents an attractive method for easy, accurate and rapid screening for 2GTKI-resistant mutations impacting on TKI selection, although ddPCR cannot identify compound mutations.

Lessons Learned from the Development and Roll-Out of the rVSVΔG-ZEBOV-GP Zaire ebolavirus Vaccine to Inform Marburg Virus and Sudan ebolavirus Vaccines.

This review describes key aspects of the development of the rVSVΔG-ZEBOV-GP Ebola vaccine and key activities which are continuing to further expand our knowledge of the product. Extensive partnerships and innovative approaches were used to address the various challenges encountered during this process. The rVSVΔG-ZEBOV-GP Ebola vaccine was initially approved by the European Medicines Agency and prequalified by the World Health Organization in November 2019. It was approved by the United States Food and Drug Administration in December 2019 and approved in five African countries within 90 days of prequalification. The development resulted in the first stockpile of a registered Ebola vaccine that is available to support outbreak response. This also provides insights into how the example of rVSVΔG-ZEBOV-GP can inform the development of vaccines for Sudan ebolavirus, Marburg virus, and other emerging epidemic diseases in terms of the types of approaches and data needed to support product registration, availability, and the use of a filovirus vaccine.