Surgical outcomes in people with hemophilia A taking emicizumab prophylaxis: experience from the HAVEN 1-4 studies.

Many people with hemophilia A (PwHA) undergo surgery in their lifetime, often because of complications of their disease. Emicizumab is the first bispecific monoclonal antibody prophylactic therapy for PwHA, and its efficacy and safety have been previously demonstrated; however, there is a need to build an evidence base on the management of PwHA on emicizumab undergoing surgery. Data from the HAVEN 1-4 phase 3 clinical trials were pooled to provide a summary of all minor and major surgeries in PwHA with or without factor VIII (FVIII) inhibitors who were receiving emicizumab prophylaxis. Overall, 233 surgeries were carried out during the HAVEN 1-4 trials: 215 minor surgeries (including minor dental and joint procedures, central venous access device placement or removal, and endoscopies) in 115 PwHA (64 with FVIII inhibitors) and 18 major surgeries (including arthroplasty and synovectomy) in 18 PwHA (10 with FVIII inhibitors). Perioperative hemostatic support was at the discretion of the treating physician. Overall, the median (interquartile range [IQR]) age was 33.5 (13.0-49.0) years and the median (IQR) emicizumab exposure time before surgery was 278.0 (177.0-431.0) days. Among the 215 minor surgeries, 141 (65.6%) were managed without additional prophylactic factor concentrate, and of those, 121 (85.8%) were not associated with a postoperative bleed. The majority (15 of 18 [83.3%]) of major surgeries were managed with additional prophylactic factor concentrate. Twelve (80.0%) of these 15 surgeries were associated with no intraoperative or postoperative bleeds. The data demonstrate that minor and major surgeries can be performed safely in PwHA receiving emicizumab prophylaxis. These trials are registered at www.clinicaltrials.gov as #NCT02622321, #NCT02795767, #NCT02847637, and #NCT03020160.

Efficacy of emicizumab prophylaxis versus factor VIII prophylaxis for treatment of hemophilia A without inhibitors: network meta-analysis and sub-group analyses of the intra-patient comparison of the HAVEN 3 trial.

Objectives: To compare the efficacy of emicizumab prophylaxis with that of factor VIII (FVIII) prophylaxis in patients with hemophilia A without inhibitors using two approaches: network meta-analyses (NMA) and additional sub-group analyses from the HAVEN 3 trial.Methods: The NMA used data from trials identified using a systematic literature review and compared bleed rates in patients receiving emicizumab prophylaxis and patients receiving FVIII prophylaxis using a Bayesian, random effects generalized linear model with log link Poisson likelihood. Additional sub-groups of the HAVEN 3 trial included here were defined as patients whose dose-taking behavior met either European label or World Federation of Hemophilia guidelines. A negative binomial regression model was used to conduct an intra-patient comparison of bleed rates within the sub-groups, during treatment with FVIII prophylaxis before entering HAVEN 3 and treatment with emicizumab prophylaxis during HAVEN 3.Results: Four studies were included in the base-case NMA. Evidence showed that the total treated bleed rate was lower with emicizumab prophylaxis compared with FVIII prophylaxis (rate ratio [RR] = 0.36 [95% credible interval (CrI) = 0.13-0.95]). Similar associations were observed in sensitivity analyses. The additional HAVEN 3 analyses also showed lower rates of treated bleeds with emicizumab prophylaxis than with FVIII prophylaxis (RRs [95% confidence interval (CI)] = 0.380 [0.186-0.790] and 0.472 [0.258-0.866] in two sub-groups). These results confirm the original HAVEN 3 intra-patient comparison findings.Conclusions: Combined findings from NMA and additional sub-group analyses of HAVEN 3 support the superiority of emicizumab prophylaxis over FVIII prophylaxis in patients with hemophilia A without inhibitors.

Improved Behavior and Neuropsychological Function in Children With ROHHAD After High-Dose Cyclophosphamide.

Rapid-onset obesity with hypothalamic dysfunction, hypoventilation, and autonomic dysregulation (ROHHAD) is a rare, generally progressive, and potentially fatal syndrome of unclear etiology. The syndrome is characterized by normal development followed by a sudden, rapid hyperphagic weight gain beginning during the preschool period, hypothalamic dysfunction, and central hypoventilation, and is often accompanied by personality changes and developmental regression, leading to substantial morbidity and mortality. We describe 2 children who had symptomatic and neuropsychological improvement after high-dose cyclophosphamide treatment. Our experience supports an autoimmune pathogenesis and provides the first neuropsychological profile of patients with rapid-onset obesity with hypothalamic dysfunction, hypoventilation, and autonomic dysregulation.

C/EBPß regulates sensitivity to bortezomib in prostate cancer cells by inducing REDD1 and autophagosome-lysosome fusion.

The purpose of this study was to ascertain the mechanisms by which advanced prostate cancer cells resist bortezomib therapy. Several independent studies have shown that cells are protected from proteasome inhibition by increased autophagic activity. We investigated whether C/EBPß, a transcription factor involved in the control of autophagic gene expression, regulates resistance to proteasome inhibition. In PC3 cells over-expressing C/EBPß, turnover of autophagic substrates and expression of core autophagy genes were increased. Conversely, C/EBPß knockdown suppressed autophagosome-lysosome fusion. We also found that C/EBPß knockdown suppressed REDD1 expression to delay early autophagy, an effect rescued by exogenous REDD1. Cells with suppressed C/EBPß levels showed delayed autophagy activation upon bortezomib treatment. Knockdown of C/EBPß sensitized PC3 cells to bortezomib, and blockade of autophagy by chloroquine did not further increase cell death in cells expressing shRNA targeting C/EBPß. Lastly, we observed a decreased growth of PC3 cells and xenografts with C/EBPß knockdown and such xenografts were sensitized to bortezomib treatment. Our results demonstrate that C/EBPß is a critical effector of autophagy via regulation of autolysosome formation and promotes resistance to proteasome inhibitor treatment by increasing autophagy.

Loss of IKKß but Not NF-κB p65 Skews Differentiation towards Myeloid over Erythroid Commitment and Increases Myeloid Progenitor Self-Renewal and Functional Long-Term Hematopoietic Stem Cells.

NF-κB is an important regulator of both differentiation and function of lineage-committed hematopoietic cells. Targeted deletion of IκB kinase (IKK) ß results in altered cytokine signaling and marked neutrophilia. To investigate the role of IKKß in regulation of hematopoiesis, we employed Mx1-Cre mediated IKKß conditional knockout mice. As previously reported, deletion of IKKß in hematopoietic cells results in neutrophilia, and we now also noted decreased monocytes and modest anemia. Granulocyte-macrophage progenitors (GMPs) accumulated markedly in bone marrow of IKKß deleted mice whereas the proportion and number of megakaryocyte-erythrocyte progenitors (MEP) decreased. Accordingly, we found a significantly reduced frequency of proerythroblasts and basophilic and polychromatic erythroblasts, and IKKß-deficient bone marrow cells yielded a significantly decreased number of BFU-E compared to wild type. These changes are associated with elevated expression of C/EBPα, Gfi1, and PU.1 and diminished Gata1, Klf1, and SCL/Tal1 in IKKß deficient Lineage-Sca1+c-Kit+ (LSK) cells. In contrast, no effect on erythropoiesis or expression of lineage-related transcription factors was found in marrow lacking NF-κB p65. Bone marrow from IKKß knockout mice has elevated numbers of phenotypic long and short term hematopoietic stem cells (HSC). A similar increase was observed when IKKß was deleted after marrow transplantation into a wild type host, indicating cell autonomous expansion. Myeloid progenitors from IKKß- but not p65-deleted mice demonstrate increased serial replating in colony-forming assays, indicating increased cell autonomous self-renewal capacity. In addition, in a competitive repopulation assay deletion of IKKß resulted in a stable advantage of bone marrow derived from IKKß knockout mice. In summary, loss of IKKß resulted in significant effects on hematopoiesis not seen upon NF-κB p65 deletion. These include increased myeloid and reduced erythroid transcription factors, skewing differentiation towards myeloid over erythroid differentiation, increased progenitor self-renewal, and increased number of functional long term HSCs. These data inform ongoing efforts to develop IKK inhibitors for clinical use.

Patterns of failure after involved field radiation therapy for pediatric and young adult Hodgkin lymphoma.

BACKGROUND: Involved field radiation therapy (IFRT) is integral in curative therapy for Hodgkin lymphoma (HL), although primarily used in patients with intermediate/high-risk HL. We present failure patterns and clinical outcomes in a cohort of pediatric and young adult patients with HL treated with IFRT at the Johns Hopkins Hospital. PROCEDURE: Patients ≤40 years old with intermediate/high-risk HL who received chemotherapy and IFRT from 1997 to 2012 were included in this retrospective analysis. Patients were evaluated for failure patterns, overall survival (OS), and event-free survival (EFS) using Kaplan-Meier curves, descriptive statistics, and Cox proportional hazard regressions. RESULTS: We reviewed 74 patients (45 pediatric and 29 young adult) with a median follow-up of 4.4 years. The mean age at diagnosis was 21.4 years. Patients received a median of 29.75 Gy of IFRT (range 15-39.6 Gy). The majority of pediatric patients received ABVE-PC chemotherapy (n = 25) and <30 Gy of radiation (n = 33) while most young adults received ABVD chemotherapy (n = 24) and ≥30 Gy (n = 25). Estimated 5-year OS and EFS were 96% and 81%, respectively. Thirteen patients had recurrence; eight were pediatric. Distant relapse alone comprised 83% of failures in patients receiving ≥30 Gy. Of the seven patients who received <30 Gy and had recurrence, six had local failure as a component of their recurrence. Caucasian race (P = 0.02) and nodular sclerosing histology (P = 0.01) predicted for increased EFS. Late effects were minimal and all deaths (n = 4) were from HL. CONCLUSIONS: In this series, pediatric and young adult patients were treated with differing chemoradiation and had distinct recurrence patterns.

A novel chordoma xenograft allows in vivo drug testing and reveals the importance of NF-κB signaling in chordoma biology.

Chordoma is a rare primary bone malignancy that arises in the skull base, spine and sacrum and originates from remnants of the notochord. These tumors are typically resistant to conventional chemotherapy, and to date there are no FDA-approved agents to treat chordoma. The lack of in vivo models of chordoma has impeded the development of new therapies for this tumor. Primary tumor from a sacral chordoma was xenografted into NOD/SCID/IL-2R γ-null mice. The xenograft is serially transplantable and was characterized by both gene expression analysis and whole genome SNP genotyping. The NIH Chemical Genomics Center performed high-throughput screening of 2,816 compounds using two established chordoma cell lines, U-CH1 and U-CH2B. The screen yielded several compounds that showed activity and two, sunitinib and bortezomib, were tested in the xenograft. Both agents slowed the growth of the xenograft tumor. Sensitivity to an inhibitor of IκB, as well as inhibition of an NF-κB gene expression signature demonstrated the importance of NF-κB signaling for chordoma growth. This serially transplantable chordoma xenograft is thus a practical model to study chordomas and perform in vivo preclinical drug testing.

C/EBPα dysregulation in AML and ALL.

The transcription factor CCAAT/enhancer binding protein a (C/EBPα) is a critical regulator of myeloid development, directing granulocyte, and monocyte differentiation. As such, it is dysregulated in more than half of patients with acute myeloid leukemia (AML). C/EBPα expression is suppressed as result of common leukemia-associated genetic and epigenetic alterations such as AML1-ETO, BCR-ABL, FLT3-ITD, or CEBPA promoter methylation. In addition, 10-15% of patients with AML with intermediate risk cytogenetics are characterized by mutations of the CEBPA gene. Two classes of mutations are described. N-terminal changes result in expression of a truncated dominant negative C/EBPαp30 isoform. C-terminal mutations are in-frame insertions or deletions resulting in alteration of the leucine zipper preventing dimerization and DNA binding. Often, patients carry both N- and C-terminal mutations each affecting a different allele, and a mouse model recapitulates the human phenotype. Patients with mutated CEBPA AML comprise a clinically distinct group with favorable outcome consistently seen in patients with biallelic mutations. In addition, C/EBP family members are aberrantly expressing from the immunoglobulin heavy chain locus in 2% of pre-B ALLs. This review summarizes the normal hematopoietic developmental pathways regulated by C/EBPα and discusses the molecular pathways involved in mutated CEBPA AML and ALL.

C/EBPα, C/EBPα oncoproteins, or C/EBPß preferentially bind NF-κB p50 compared with p65, focusing therapeutic targeting on the C/EBP:p50 interaction.

Canonical nuclear factor kappaB (NF-κB) activation signals stimulate nuclear translocation of p50:p65, replacing inhibitory p50:p50 with activating complexes on chromatin. C/EBP interaction with p50 homodimers provides an alternative pathway for NF-κB target gene activation, and interaction with p50:p65 may enhance gene activation. We previously found that C/EBPα cooperates with p50, but not p65, to induce Bcl-2 transcription and that C/EBPα induces Nfkb1/p50, but not RelA/p65, transcription. Using p50 and p65 variants containing the FLAG epitope at their N- or C-termini, we now show that C/EBPα, C/EBPα myeloid oncoproteins, or the LAP1, LAP2, or LIP isoforms of C/EBPß have markedly higher affinity for p50 than for p65. Deletion of the p65 transactivation domain did not increase p65 affinity for C/EBPs, suggesting that unique residues in p50 account for specificity, and clustered mutation of HSDL in the "p50 insert" lacking in p65 weakens interaction. Also, in contrast to Nfkb1 gene deletion, absence of the RelA gene does not reduce Bcl-2 or Cebpa RNA in unstimulated cells or prevent interaction of C/EBPα with the Bcl-2 promoter. Saturating mutagenesis of the C/EBPα basic region identifies R300 and nearby residues, identical in C/EBPß, as critical for interaction with p50. These findings support the conclusion that C/EBPs activate NF-κB target genes via contact with p50 even in the absence of canonical NF-κB activation and indicate that targeting C/EBP:p50 rather than C/EBP:p65 interaction in the nucleus will prove effective for inflammatory or malignant conditions, alone or synergistically with agents acting in the cytoplasm to reduce canonical NF-κB activation.

C/EBPα and C/EBPα oncoproteins regulate nfkb1 and displace histone deacetylases from NF-κB p50 homodimers to induce NF-κB target genes.

Mutated CEBPA defines a subgroup of acute myeloid leukemia (AML). We have previously shown that C/EBPα or its AML mutants synergize with NF-κB p50 to activate antiapoptotic genes, including BCL2 and FLIP. Furthermore, p50 binds and activates the CEBPA gene in myeloid cells. We now report that C/EBPα or C/EBPα leucine zipper AML mutants bind in vivo to the nfkb1 (p50) promoter and induce its expression even in the presence of cycloheximide. Induction of p50 by C/EBPα depends on 2 conserved κB sites in the nfkb1 promoter. C/EBPα did not induce p65 expression. Thus, C/EBPα and p50 reciprocally regulate each other's expression, establishing a positive feedback relationship. Although p50 homodimers inhibit transcription, C/EBPα and p50 synergistically activate antiapoptotic genes. ChIP analysis showed that C/EBPα diminishes the occupation of histone deacetylase 1 (HDAC1) or HDAC3 on the endogenous FLIP promoter but not in mice lacking p50. Coimmunoprecipitation confirmed that C/EBPα, its AML variants, or C/EBPß disrupt interaction between p50 and HDACs dependent on the C/EBP basic region. These findings suggest that C/EBPs displace HDACs from p50 homodimers bound to antiapoptotic genes, contributing to NF-κB dysregulation in leukemia, and that the C/EBPα:p50 complex is a potential therapeutic target.

Cyclophosphamide for rapid-onset obesity, hypothalamic dysfunction, hypoventilation, and autonomic dysregulation syndrome.

Patients with rapid-onset obesity, hypothalamic dysfunction, hypoventilation, autonomic dysregulation, and neural crest tumor syndrome have poor long-term outcomes. We report a patient who was treated successfully with high-dose cyclophosphamide immunoablation. This experience offers a novel therapeutic approach and an indirect insight into the underlying pathogenesis of this syndrome.

C/EBPalpha binds and activates the PU.1 distal enhancer to induce monocyte lineage commitment.

The PU.1 gene contains a 237-base pair distal enhancer located 14 kilobases upstream of its promoter. We have identified 2 sites within the PU.1 enhancer that strongly bind C/EBPalpha in a gel shift assay, and interaction with endogenous C/EBPalpha was confirmed by chromatin immunoprecipitation. Mutation of these DNA elements reduced activity of a distal enhancer-promoter construct 2- or 5-fold in a myeloid cell line, while mutation of a weaker C/EBPalpha-binding site located in the promoter minimally reduced activity in this context. These findings strengthen the link between C/EBPalpha and PU.1 expression. Reduction of C/EBPalpha activity in cases of acute myeloid leukemia may therefore contribute to transformation by reducing PU.1 levels. In addition, induction of PU.1 by C/EBPalpha during normal hematopoiesis may contribute to stem cell commitment to the myeloid lineages and further commitment to monopoiesis. Consistent with a requirement for C/EBPalpha induction of PU.1 during myeloid development, we demonstrate that C/EBPalpha induces monocytic development when expressed in PU.1(+/+), PU.1(+/-), or PU.1(+/kd) marrow myeloid progenitors but induces granulocyte lineage commitment in PU.1(kd/kd) cells lacking the PU.1 distal enhancer and does not induce either lineage in PU.1(-/-) cells.

Thromboembolic events in children and young adults with pediatric sarcoma.

PURPOSE: Adults with malignancy are at increased risk for venous thromboembolic events (TEs). However, data in children and young adults with cancer are limited. PATIENTS AND METHODS: To determine the risk and clinical features of TEs in children and young adults with sarcoma, we reviewed records on 122 consecutive patients with sarcoma treated from October 1980 to July 2002. RESULTS: Twenty-three TEs were diagnosed in 19 of 122 (16%; 95% CI, 10% to 23%) patients. Prevalence by diagnosis was Ewing sarcoma, eight of 61 (13%); osteosarcoma, two of 20 (10%); rhabdomyosarcoma, four of 26 (15%); and other sarcomas, five of 15 (33%). TEs developed in 23% of patients with metastases at presentation versus 10% with localized disease (odds ratio, 2.59; 95% CI, 0.9 to 7.1; P < .06). Fifty-three percent of patients with thrombosis had a clot at presentation. A lupus anticoagulant was detected in four of five evaluated patients. There was a single fatality due to pulmonary embolism. Patients who were diagnosed with cancer after 1993 had a higher rate of TE (7% v 23%; P < .015). Of the 23 events, 43% were asymptomatic. Main sites of thromboses were deep veins of the extremities (10 of 23; 43%), pulmonary embolism (five of 23; 22%), and the inferior vena cava (four of 23; 17%). TEs were associated with tumor compression in eight of 23 (35%) and with venous catheters in three of 23 (13%). CONCLUSION: Thromboembolism is common in pediatric patients with sarcomas. Thromboses are detected frequently around the time of oncologic presentation, may be asymptomatic, and seem to be associated with a higher disease burden. Children and young adults with sarcoma should be monitored closely for thrombosis.

CCAAT/enhancer binding protein alpha (C/EBPalpha) and C/EBPalpha myeloid oncoproteins induce bcl-2 via interaction of their basic regions with nuclear factor-kappaB p50.

The CEBPA gene is mutated in 10% of acute myeloid leukemia (AML) cases. We find that CEBPA and Bcl-2 RNA levels correlate highly in low-risk human AMLs, suggesting that inhibition of apoptosis via induction of bcl-2 by CCAAT/enhancer binding protein alpha (C/EBPalpha) or its mutant variants contributes to transformation. C/EBPalphap30, lacking a NH2-terminal transactivation domain, or C/EBPalphaLZ, carrying in-frame mutations in the leucine zipper that prevent DNA binding, induced bcl-2 in hematopoietic cell lines, and C/EBPalpha induced bcl-2 in normal murine myeloid progenitors and in the splenocytes of H2K-C/EBPalpha-Emu transgenic mice. C/EBPalpha protected Ba/F3 cells from apoptosis on interleukin-3 withdrawal but not if bcl-2 was knocked down. Remarkably, C/EBPalphaLZ oncoproteins activated the bcl-2 P2 promoter despite lack of DNA binding, and C/EBPalphap30 also activated the promoter. C/EBPalpha and the C/EBPalpha oncoproteins cooperated with nuclear factor-kappaB (NF-kappaB) p50, but not p65, to induce bcl-2 transcription. Endogenous C/EBPalpha preferentially coimmunoprecipitated with p50 versus p65 in myeloid cell extracts. Mutation of residues 297 to 302 in the C/EBPalpha basic region prevented induction of endogenous bcl-2 or the bcl-2 promoter and interaction with p50 but not p65. These findings suggest that C/EBPalpha or its mutant variants tether to a subset of NF-kappaB target genes, including Bcl-2, via p50 to facilitate gene activation and offer an explanation for preferential in-frame rather than out-of-frame mutation of the leucine zipper with sparing of the basic region in C/EBPalphaLZ oncoproteins. Targeting interaction between C/EBPalpha basic region and NF-kappaB p50 may contribute to the therapy of AML and other malignancies expressing C/EBPs.