In vivo clonal tracking reveals evidence of haemangioblast and haematomesoblast contribution to yolk sac haematopoiesis.

During embryogenesis, haematopoietic and endothelial lineages emerge closely in time and space. It is thought that the first blood and endothelium derive from a common clonal ancestor, the haemangioblast. However, investigation of candidate haemangioblasts in vitro revealed the capacity for mesenchymal differentiation, a feature more compatible with an earlier mesodermal precursor. To date, no evidence for an in vivo haemangioblast has been discovered. Using single cell RNA-Sequencing and in vivo cellular barcoding, we have unravelled the ancestral relationships that give rise to the haematopoietic lineages of the yolk sac, the endothelium, and the mesenchyme. We show that the mesodermal derivatives of the yolk sac are produced by three distinct precursors with dual-lineage outcomes: the haemangioblast, the mesenchymoangioblast, and a previously undescribed cell type: the haematomesoblast. Between E5.5 and E7.5, this trio of precursors seeds haematopoietic, endothelial, and mesenchymal trajectories.

BRD4 bimodal binding at promoters and drug-induced displacement at Pol II pause sites associates with I-BET sensitivity.

BACKGROUND: Deregulated transcription is a major driver of diseases such as cancer. Bromodomain and extra-terminal (BET) proteins (BRD2, BRD3, BRD4 and BRDT) are chromatin readers essential for maintaining proper gene transcription by specifically binding acetylated lysine residues. Targeted displacement of BET proteins from chromatin, using BET inhibitors (I-BETs), is a promising therapy, especially for acute myeloid leukemia (AML), and evaluation of resistance mechanisms is necessary to optimize the clinical efficacy of these drugs. RESULTS: To uncover mechanisms of intrinsic I-BET resistance, we quantified chromatin binding and displacement for BRD2, BRD3 and BRD4 after dose response treatment with I-BET151, in sensitive and resistant in vitro models of leukemia, and mapped BET proteins/I-BET interactions genome wide using antibody- and compound-affinity capture methods followed by deep sequencing. The genome-wide map of BET proteins sensitivity to I-BET revealed a bimodal pattern of binding flanking transcription start sites (TSSs), in which drug-mediated displacement from chromatin primarily affects BRD4 downstream of the TSS and prolongs the pausing of RNA Pol II. Correlation of BRD4 binding and drug-mediated displacement at RNA Pol II pause sites with gene expression revealed a differential behavior of sensitive and resistant tumor cells to I-BET and identified a BRD4 signature at promoters of sensitive coding and non-coding genes. CONCLUSIONS: We provide evidence that I-BET-induced shift of Pol II pausing at promoters via displacement of BRD4 is a determinant of intrinsic I-BET sensitivity. This finding may guide pharmacological treatment to enhance the clinical utility of such targeted therapies in AML and potentially other BET proteins-driven diseases.

Prediction and monitoring of relapse in stage III melanoma using circulating tumor DNA.

BACKGROUND: The advent of effective adjuvant therapies for patients with resected melanoma has highlighted the need to stratify patients based on risk of relapse given the cost and toxicities associated with treatment. Here we assessed circulating tumor DNA (ctDNA) to predict and monitor relapse in resected stage III melanoma. PATIENTS AND METHODS: Somatic mutations were identified in 99/133 (74%) patients through tumor tissue sequencing. Personalized droplet digital PCR (ddPCR) assays were used to detect known mutations in 315 prospectively collected plasma samples from mutation-positive patients. External validation was performed in a prospective independent cohort (n = 29). RESULTS: ctDNA was detected in 37 of 99 (37%) individuals. In 81 patients who did not receive adjuvant therapy, 90% of patients with ctDNA detected at baseline and 100% of patients with ctDNA detected at the postoperative timepoint relapsed at a median follow up of 20 months. ctDNA detection predicted patients at high risk of relapse at baseline [relapse-free survival (RFS) hazard ratio (HR) 2.9; 95% confidence interval (CI) 1.5-5.6; P = 0.002] and postoperatively (HR 10; 95% CI 4.3-24; P < 0.001). ctDNA detection at baseline [HR 2.9; 95% CI 1.3-5.7; P = 0.003 and postoperatively (HR 11; 95% CI 4.3-27; P < 0.001] was also associated with inferior distant metastasis-free survival (DMFS). These findings were validated in the independent cohort. ctDNA detection remained an independent predictor of RFS and DMFS in multivariate analyses after adjustment for disease stage and BRAF mutation status. CONCLUSION: Baseline and postoperative ctDNA detection in two independent prospective cohorts identified stage III melanoma patients at highest risk of relapse and has potential to inform adjuvant therapy decisions.

Recurrent mutations, including NPM1c, activate a BRD4-dependent core transcriptional program in acute myeloid leukemia.

Recent evidence suggests that inhibition of bromodomain and extra-terminal (BET) epigenetic readers may have clinical utility against acute myeloid leukemia (AML). Here we validate this hypothesis, demonstrating the efficacy of the BET inhibitor I-BET151 across a variety of AML subtypes driven by disparate mutations. We demonstrate that a common 'core' transcriptional program, which is HOX gene independent, is downregulated in AML and underlies sensitivity to I-BET treatment. This program is enriched for genes that contain 'super-enhancers', recently described regulatory elements postulated to control key oncogenic driver genes. Moreover, our program can independently classify AML patients into distinct cytogenetic and molecular subgroups, suggesting that it contains biomarkers of sensitivity and response. We focus AML with mutations of the Nucleophosmin gene (NPM1) and show evidence to suggest that wild-type NPM1 has an inhibitory influence on BRD4 that is relieved upon NPM1c mutation and cytosplasmic dislocation. This leads to the upregulation of the core transcriptional program facilitating leukemia development. This program is abrogated by I-BET therapy and by nuclear restoration of NPM1. Finally, we demonstrate the efficacy of I-BET151 in a unique murine model and in primary patient samples of NPM1c AML. Taken together, our data support the use of BET inhibitors in clinical trials in AML.

BET protein inhibition shows efficacy against JAK2V617F-driven neoplasms.

Small molecule inhibition of the BET family of proteins, which bind acetylated lysines within histones, has been shown to have a marked therapeutic benefit in pre-clinical models of mixed lineage leukemia (MLL) fusion protein-driven leukemias. Here, we report that I-BET151, a highly specific BET family bromodomain inhibitor, leads to growth inhibition in a human erythroleukemic (HEL) cell line as well as in erythroid precursors isolated from polycythemia vera patients. One of the genes most highly downregulated by I-BET151 was LMO2, an important oncogenic regulator of hematopoietic stem cell development and erythropoiesis. We previously reported that LMO2 transcription is dependent upon Janus kinase 2 (JAK2) kinase activity in HEL cells. Here, we show that the transcriptional changes induced by a JAK2 inhibitor (TG101209) and I-BET151 in HEL cells are significantly over-lapping, suggesting a common pathway of action. We generated JAK2 inhibitor resistant HEL cells and showed that these retain sensitivity to I-BET151. These data highlight I-BET151 as a potential alternative treatment against myeloproliferative neoplasms driven by constitutively active JAK2 kinase.

Knowledge, perception and practices of healthcare professionals at tertiary level hospitals in Kingston, Jamaica, regarding neonatal pain management.

OBJECTIVE: To determine knowledge, perception and practices of healthcare professionals at tertiary level hospitals in Kingston, Jamaica, regarding neonatal pain management. DESIGN AND METHODS: Physicians and nurses actively involved in providing neonatal care at three tertiary level hospitals were invited to participate. A 21-item self-administered questionnaire was used to obtain information on knowledge, perception and practice of neonatal pain management. Descriptive analyses were performed. RESULTS: A total of 147 healthcare workers participated giving a response rate of 85%. Male to female ratio was 1: 4.4. Nurses accounted for 76 (52%) of the respondents while 70 (48%) were physicians. Seventy-three (50%) individuals were unaware of the degree of pain neonates were capable of experiencing and only 38 (27%) knew that premature infants were capable of feeling pain. One hundred and four (71%) respondents were able to identify physiological markers of pain and most respondents were able to discriminate between painful and non-painful procedures. However, 100 (68%) respondents rarely prescribed analgesia for procedures previously rated as painful. Seventy-one (51%) respondents admitted to not using analgesia for alleviating procedural pain in neonates. Twenty-five (18%) individuals thought that the procedure was too short to require analgesic support while 41 (30%) stated that medication was not usually prescribed for procedural pain. Physician scores were significantly higher than those attained by nurses for knowledge (p = 0.003) and for pain perception (p = 0.001) but no significant differences were noted for practice (p = 0.18). CONCLUSION: There is an overwhelming deficiency in the knowledge, perception and practice of neonatal pain management at tertiary level institutions in Kingston, Jamaica. There is the urgent need for the education of health professionals on neonatal pain management. This will in turn facilitate change in perception and eventually, along with the institution of local policies and protocols, influence practice.

Linezolid-induced dyserythropoiesis: chloramphenicol toxicity revisited.

We report here two cases of dyserythropoietic anaemia associated with long-term linezolid use that share striking similarities to chloramphenicol-associated myelotoxicity.

Transjugular liver biopsy is a safe and effective intervention to guide management for patients with a congenital bleeding disorder infected with hepatitis C.

The prevalence of hepatitis C virus (HCV) infection in adult patients with a congenital bleeding disorder (CBD) approaches 95% and is a major cause of morbidity and mortality. Histological examination of the liver remains the cornerstone of management decisions in patients without a CBD. The reluctance to perform liver biopsies in patients with a CBD has been a major limitation in the management of these patients. We are currently the only haemophilia centre in Australasia performing liver biopsies in patients with a CBD for the purpose of guiding prognostic and therapeutic decisions. We report here the results of our centre's experience with transjugular liver biopsy (TJLB) in patients with a CBD. An adequate specimen for histological assessment was attained from all of the patients. There were no major complications recorded. Patients were hospitalized for < or = 48 h for haemostasis prophylaxis. The diagnostic specimen obtained from patients was integral in guiding their future management. We suggest that with a coordinated multidisciplinary approach, TJLB can be performed in patients with a CBD.

Successful mobilization of peripheral blood stem cells using recombinant human stem cell factor in heavily pretreated patients who have failed a previous attempt with a granulocyte colony-stimulating factor-based regimen.

To assess the efficacy of recombinant human stem cell factor (rHuSCF), 48 patients who had failed to mobilize >2.0 x 10(6) CD34+ cells/kg with granulocyte colony-stimulating factor (G-CSF) (10 microg/kg twice daily) with, or without, concomitant chemotherapy (G-CSF-based regimen), were remobilized with the addition of rHuSCF (20 microg/kg/day). In all, 18/48 (38%) achieved a total of >2.0 x 10(6) CD34+ cells/kg with the second rHuSCF-based mobilisation alone and 29/48 (60%) achieved a cumulative total of >2.0 x 10(6) CD34+ cells/kg following remobilization. Inclusion of chemotherapy in the mobilization regimen resulted in a higher yield of CD34+ cells/kg for both the initial G-CSF-based and subsequent rHuSCF-based regimens (0.90 vs 0.54, P < 0.01 and 2.36 vs 1.34, P < 0.01, respectively). The total peripheral blood stem cells PBSC collected from the G-CSF-based regimen, performance status, baseline platelet count and albumin were significantly associated with successful remobilization. Patients with multiple myeloma were also more likely to successfully remobilize. There was no threshold of total collected from the failed G-CSF-based regimen below which successful remobilization with the rHuSCF-based regimen was not possible. We therefore propose a predictive model [PBSC expected = 0.6+(G-CSF-based total collection)+2 (rHuSCF-based day 1 collection)] to calculate the cumulative total of PBSC expected following a maximum of five leukaphereses. This algorithm may permit the early identification of patients who are unlikely to achieve sufficient PBSC for transplantation and allow physicians to direct the resources involved in PBSC collection in a more appropriate and economical manner.

Efficacy of donor vaccination before hematopoietic cell transplantation and recipient vaccination both before and early after transplantation.

Allogeneic hematopoietic cell transplantation is followed by humoral immunodeficiency. We evaluated whether antibody levels can be improved by recipient vaccination on day -1 and 50 and whether the levels can be further improved by donor vaccination on day -20. A total of 85 patients were randomized or assigned to one of the following strategies of immunization with Streptococcus pneumoniae polysaccharides, Haemophilus influenzae polysaccharide-protein conjugate, tetanus toxoid (protein recall antigen) and hepatitis B surface antigen (protein neo-antigen): (1) donor on day -20, recipient on days -1, +50 and +365 (D(-20)R(-1,50,365)); (2) donor nil, recipient on days -1, +50 and +365 (D(N)R(-1,50,365)); or (3) donor nil, recipient on day +365 (D(N)R(365)). For H. influenzae and tetanus, IgG levels after grafting were the highest in the D(-20)R(-1,50,365) patients, intermediate in the D(N)R(-1,50,365) patients and the lowest in the D(N)R(365) patients. For S. pneumoniae and hepatitis B, antibody levels appeared to be similar in all three patient groups. The results suggest that for polysaccharide-protein conjugate antigens or protein recall antigens, recipient immunization on days -1 and 50 improves antibody levels and that donor vaccination on day -20 further improves the levels. In contrast, neither recipient immunization on days -1 and 50 nor donor immunization on day -20 appears to be efficacious for polysaccharide antigens and poorly immunogenic protein neo-antigens.

Immunity of patients surviving 20 to 30 years after allogeneic or syngeneic bone marrow transplantation.

The duration of immunodeficiency following marrow transplantation is not known. Questionnaires were used to study the infection rates in 72 patients surviving 20 to 30 years after marrow grafting. Furthermore, in 33 of the 72 patients and in 16 donors (siblings who originally donated the marrow) leukocyte subsets were assessed by flow cytometry. T-cell receptor excision circles (TRECs), markers of T cells generated de novo, were quantitated by real-time polymerase chain reaction. Immunoglobulin G(2) (IgG(2)) and antigen-specific IgG levels were determined by enzyme-linked immunosorbent assay. Infections diagnosed more than [corrected] 15 years after transplantation occurred rarely. The average rate was 0.07 infections per patient-year (one infection every 14 years), excluding respiratory tract infections, gastroenteritis, lip sores, and hepatitis C. The counts of circulating monocytes, natural killer cells, B cells, CD4 T cells, and CD8 T cells in the patients were not lower than in the donors. The counts of TREC(+) CD4 T cells in transplant recipients younger than age 18 years (at the time of transplantation) were not different from the counts in their donors. In contrast, the counts of TREC(+) CD4 T cells were lower in transplant recipients age 18 years or older, even in those with no history of clinical extensive chronic graft-versus-host disease, compared with their donors. The levels of total IgG(2) and specific IgG against Haemophilus influenzae and Streptococcus pneumoniae were similar in patients and donors. Overall, the immunity of patients surviving 20 to 30 years after transplantation is normal or near normal. Patients who received transplants in adulthood have a clinically insignificant deficiency of de novo-generated CD4 T cells, suggesting that in these patients the posttransplantation thymic insufficiency may not be fully reversible.

Factors influencing B lymphopoiesis after allogeneic hematopoietic cell transplantation.

In 93 allograft recipients, the numbers of marrow B-cell precursors on days 80 and 365 correlated with the counts of circulating B cells, suggesting that the posttransplantation B-cell deficiency is at least in part due to insufficient B lymphopoiesis. Factors that could affect B lymphopoiesis were evaluated. The number of marrow B-cell precursors on days 30 and 80 was at least 4-fold lower in patients with grade 2 to 4 acute graft-versus-host disease (GVHD) compared with patients with grade 0 to 1 acute GVHD. The number of B-cell precursors on day 365 was 18-fold lower in patients with extensive chronic GVHD compared with patients with no or limited chronic GVHD. The number of B-cell precursors was not related to CD34 cell dose, type of transplant (marrow versus blood stem cells), donor age, or patient age. It was concluded that posttransplantation B-cell deficiency results in part from inhibition of B lymphopoiesis by GVHD and/or its treatment.

Immune reconstitution after allogeneic marrow transplantation compared with blood stem cell transplantation.

Allogeneic peripheral blood stem cell grafts contain about 10 times more T and B cells than marrow grafts. Because these cells may survive in transplant recipients for a long time, recipients of blood stem cells may be less immunocompromised than recipients of marrow. Immune reconstitution was studied in 115 patients randomly assigned to receive either allogeneic marrow or filgrastim-mobilized blood stem cell transplantation. Between day 30 and 365 after transplantation, counts of most lymphocyte subsets were higher in the blood stem cell recipients. The difference was most striking for CD4 T cells (about 4-fold higher counts for CD45RA(high) CD4 T cells and about 2-fold higher counts for CD45RA(low/-)CD4 T cells; P <.05). On assessment using phytohemagglutinin and herpesvirus antigen-stimulated proliferation, T cells in the 2 groups of patients appeared equally functional. Median serum IgG levels were similar in the 2 groups. The rate of definite infections after engraftment was 1.7-fold higher in marrow recipients (P =.001). The rate of severe (inpatient treatment required) definite infections after engraftment was 2.4-fold higher in marrow recipients (P =.002). The difference in the rates of definite infections was greatest for fungal infections, intermediate for bacterial infections, and lowest for viral infections. Death associated with a fungal or bacterial infection occurred between day 30 and day 365 after transplantation in 9 marrow recipients and no blood stem cell recipients (P =.008). In conclusion, blood stem cell recipients have higher lymphocyte-subset counts and this appears to result in fewer infections. (Blood. 2001;97:3380-3389)

Low B-cell and monocyte counts on day 80 are associated with high infection rates between days 100 and 365 after allogeneic marrow transplantation.

To ascertain which mononuclear cell subset deficiency plays a role in the marrow transplant recipient's susceptibility to infections, mononuclear cell subset counts were prospectively determined in 108 patients on day 80. Infections occurring between day 100 and 365 were recorded by an investigator blinded to the subset counts. In univariate analyses, the counts of the following subsets showed a significant inverse correlation with infection rates: total B cells, IgD(+) B cells, IgD(-) B cells, total CD4 T cells, CD28(+) CD4 T cells, CD28(-) CD4 T cells, CD45RA(low/-) CD4 T cells and monocytes. In multivariate analyses, the counts of the following subsets remained significantly inversely correlated with the infection rates: total B cells (P =.0004) and monocytes (P =.009). CD28(-) CD8 T-cell counts showed no correlation with infection rates. In conclusion, the susceptibility of patients to infections late posttransplant may be due in part to the slow reconstitution of B cells and monocytes.

B-cell-autonomous somatic mutation deficit following bone marrow transplant.

Hematopoietic stem cell transplantation is characterized by a prolonged period of humoral immunodeficiency. We have previously shown that the deficiencies are probably not due to the failure to utilize the appropriate V regions in the pre-immune repertoire. However, a striking observation, which correlated with the absence of immunoglobulin IgD(-) cells and was consistent with a defect in antigen-driven responses, was that rearrangements in bone marrow transplant (BMT) recipients exhibited much less somatic mutation than did rearrangements obtained from healthy subjects. In this paper, we present evidence suggesting that naive B cells obtained from BMT recipients lack the capacity to accumulate somatic mutations in a T-cell-dependent manner compared with healthy subjects. This appears to be a B-cell-autonomous deficit because T cells from some patients, which were not able to support the accumulation of mutations in autologous naive B cells, were able to support accumulation of mutations in heterologous healthy-subject naive B cells.