Reactivation of human herpesvirus 6 in pediatric allogeneic hematopoietic stem cell transplant recipients.

BACKGROUND: Reactivation of human herpesvirus 6 (HHV-6) occurs in 30%-50% of patients (pts) who receive allogeneic (allo) hematopoietic stem cell transplant (HCT). However, the recommendation for post-transplant HHV-6 monitoring and treatment in pediatric pts is not well established. METHODS: HHV-6 incidence rates and the clinical outcomes were reported for 139 pediatric pts (≤18 years) undergoing first allo-HCT at City of Hope from July 2011 to July 2017, for whom HHV-6 was monitored weekly throughout HCT hospitalization. For 57 pediatric pts, who underwent first HCT from January 2009 to July 2011, HHV-6 was tested as clinically indicated and only rates of HHV-6 viremia were collected. RESULTS: From July 2011 to July 2017, HHV-6 was detected in 88/139 pts (63%). The frequency of HHV-6 viremia was associated with malignant diagnoses, myeloablative conditioning, and cord blood HCT. Treatment with antiviral agents was offered to symptomatic pts with a higher viral load (VL), for whom the time to VL clearance was longer and the frequency of subsequent recurrences was higher. Pts with a lower VL cleared HHV-6 without treatment. HHV-6 viremia was associated with a higher frequency of grade II-IV acute graft-versus-host disease (GVHD) (P = .022), but did not affect overall survival (OS), disease-free survival (DFS), non-relapsed mortality (NRM), myeloid, or platelet (Plt) engraftment. CONCLUSIONS: HHV-6 weekly screening is not necessary for all HCT pts but may be considered for high-risk pts with malignant diagnoses undergoing cord blood HCT; otherwise, HHV-6 should be tested as clinically indicated. Only symptomatic pts (especially with a high VL > 25 000) could benefit from treatment. HHV-6 viremia at the time of initiation and administration of the conditioning regimen cleared promptly without the need to augment the transplant process.

Long-Term Follow-up of High-Dose Chemotherapy with Autologous Stem Cell Transplantation in Children and Young Adults with Metastatic or Relapsed Ewing Sarcoma: A Single-Institution Experience.

Forty-seven patients with metastatic disease at diagnosis or recurrent Ewing sarcoma (EWS) received high-dose chemotherapy (HDC) followed by tandem (n = 20, from February 13, 1997, to October 24, 2002) or single (n = 27, from October 1, 2004, to September 5, 2018) autologous hematopoietic stem cell transplantation (ASCT). To our knowledge, this is the largest single-institution study with sustained long-term follow-up exceeding 10 years. All patients who underwent single ASCT received a novel conditioning regimen with busulfan, melphalan, and topotecan. The overall survival (OS) and disease-free survival (DFS) were 46% and 37% at 10 years and 42% and 37% at 15 years, respectively. Disease status at transplant and the time to disease relapse prior to ASCT were identified as important prognostic factors in OS, DFS, and risk of relapse. At 10 years, patients who underwent transplantation in first complete response (1CR) had an excellent outcome (OS 78%), patients in 1CR/second complete response (2CR)/first partial response (1PR) had an OS of 66%, and patients at third or more complete response, second or more partial response, or advanced disease had an OS of 26%. Ten-year OS for patients without a history of relapse, with late relapse (≥2 years from diagnosis), or with early relapse (<2 years from diagnosis) was 75%, 50%, and 18%, respectively. Selected patients in 1CR, 2CR, 1PR, and with late relapse had excellent, sustained 10- and 15-year OS and DFS.

Treatment of post-transplant lymphoproliferative disorder (PTLD) in a heart transplant recipient with chimeric antigen receptor T-cell therapy.

Post-transplant lymphoproliferative disorders (PTLD) are a group of lesions that can complicate solid organ or hematopoietic stem cell transplantation and are often associated with Epstein-Barr virus (EBV). The treatment of PTLD is dependent on the type of lesion and includes a wide range of therapies, but chimeric antigen receptor (CAR) T-cell therapy has not previously been reported as a treatment option for PTLD. We present a patient who developed refractory PTLD in her right retroperitoneum, right inguinal and iliac chains, and right axillary region shortly after heart transplantation and was treated with CAR T-cell therapy. She could not tolerate complete discontinuation of immunosuppression due to the risk of rejection of a life-supporting graft. The patient's PTLD responded to CAR T-cell therapy, and her heart was monitored throughout the treatment course without any signs of rejection or ventricular dysfunction. CAR T-cell therapy may be a viable treatment option in patients who develop PTLD after a solid organ transplant.

HLA Haploidentical Stem Cell Transplant with Pretransplant Immunosuppression for Patients with Sickle Cell Disease.

Allogeneic stem cell transplantation (HCT) is curative in patients with severe sickle cell disease (SCD), but a significant number of patients lack an HLA-identical sibling or matched unrelated donor. Mismatched related (haploidentical) HCT with post-transplant cyclophosphamide (PTCY) allows expansion of the donor pool but is complicated by high rates of graft failure. In this report we describe a favorable haploidentical HCT approach in a limited cohort of SCD patients with significant comorbidities. To reduce the risk of graft failure we administered the conditioning regimen of rabbit antithymocyte globulin, busulfan, and fludarabine preceded with 2 courses of pretransplant immunosuppressive therapy (PTIS) with fludarabine and dexamethasone. Graft-versus-host disease (GVHD) prophylaxis consisted of PTCY on days +3 and +4 followed by tacrolimus and mycophenolate mofetil starting on day +5. Four patients (ages 13, 19, 19, and 23 years) received T cell-replete haploidentical stem cell infusion. All patients engrafted with 99.9% to 100% donor chimerism, and all patients continued with stable engraftment at the last follow-up (5 to 11 months post-transplant). Time to neutrophil engraftment was 14 to 26 days. Two patients had high levels of donor-specific anti-HLA antibodies, which required the implementation of an antibody management protocol. This facilitated neutrophil engraftment on day +16 and day +26, respectively. One patient developed grade I acute GVHD, which resolved. Three patients developed mild, limited skin GVHD that responded to conventional immunosuppressive therapy. Human herpesvirus-6 viremia was detected in 3 patients but resolved without treatment. One patient developed asymptomatic cytomegalovirus viremia that responded appropriately to standard therapy with ganciclovir. The prompt, stable engraftment and low toxicity in the post-transplant period makes PTIS with haploidentical transplant a promising option for patients with SCD.

Neutropenia-associated ELANE mutations disrupting translation initiation produce novel neutrophil elastase isoforms.

Hereditary neutropenia is usually caused by heterozygous germline mutations in the ELANE gene encoding neutrophil elastase (NE). How mutations cause disease remains uncertain, but two hypotheses have been proposed. In one, ELANE mutations lead to mislocalization of NE. In the other, ELANE mutations disturb protein folding, inducing an unfolded protein response in the endoplasmic reticulum (ER). In this study, we describe new types of mutations that disrupt the translational start site. At first glance, they should block translation and are incompatible with either the mislocalization or misfolding hypotheses, which require mutant protein for pathogenicity. We find that start-site mutations, instead, force translation from downstream in-frame initiation codons, yielding amino-terminally truncated isoforms lacking ER-localizing (pre) and zymogen-maintaining (pro) sequences, yet retain essential catalytic residues. Patient-derived induced pluripotent stem cells recapitulate hematopoietic and molecular phenotypes. Expression of the amino-terminally deleted isoforms in vitro reduces myeloid cell clonogenic capacity. We define an internal ribosome entry site (IRES) within ELANE and demonstrate that adjacent mutations modulate IRES activity, independently of protein-coding sequence alterations. Some ELANE mutations, therefore, appear to cause neutropenia via the production of amino-terminally deleted NE isoforms rather than by altering the coding sequence of the full-length protein.

Rosette-forming glioneuronal tumor: a pineal region case with IDH1 and IDH2 mutation analyses and literature review of 43 cases.

Rosette-forming glioneuronal tumor (RGNT) of the fourth ventricle is a mixed glio-neuronal neoplasm recently codified by the World Health Organization WHO Classification of Central Nervous System (CNS) Tumors (2007). To date, 43 cases have been described in the literature; most occurring in the fourth ventricle region. We report the fourth case involving the pineal region in a 16-year-old female with signs of increased intracranial pressure (ICP). A stereotactic biopsy of the mass was followed by a debulking procedure. Both specimens revealed classic RGNT histology. The patient had stable scans 7 months post-resection. The clinical, radiological and histopathologic features of the previously described 43 cases are reviewed along with our illustrative case. Mean age of patients was 30 ± 12.8 years with 1.9:1 female to male ratio. The most common presenting signs related to increased ICP and posterior fossa involvement, including: headache (62.8%), ataxia (39.5%) and vomiting and vertigo (both 16.3%). This tumor usually presents with cystic changes (54.5%) with focal enhancement (60.9%) and hydrocephalus (43.2%). Microcalcifications and satellite lesions were common radiographic observations. All reported cases had the classic biphasic pattern. Rosenthal fibers and eosinophilic granular bodies are each present in approximately two thirds of cases. Ki-67 labeling index is consistently low (mean (%): 1.8 ± 0.75 SD). The isocitrate dehydrogenase 1 or 2 mutation found in low grade diffuse gliomas is not identified in this RGNT case. Reported outcome is nearly uniformly excellent after complete or subtotal resection. A solitary report of recurrence after 10 years and the limited experience with this entity suggest that long term follow up is advisable.

Expression profile of CREB knockdown in myeloid leukemia cells.

BACKGROUND: The cAMP Response Element Binding Protein, CREB, is a transcription factor that regulates cell proliferation, differentiation, and survival in several model systems, including neuronal and hematopoietic cells. We demonstrated that CREB is overexpressed in acute myeloid and leukemia cells compared to normal hematopoietic stem cells. CREB knockdown inhibits leukemic cell proliferation in vitro and in vivo, but does not affect long-term hematopoietic reconstitution. METHODS: To understand downstream pathways regulating CREB, we performed expression profiling with RNA from the K562 myeloid leukemia cell line transduced with CREB shRNA. RESULTS: By combining our expression data from CREB knockdown cells with prior ChIP data on CREB binding we were able to identify a list of putative CREB regulated genes. We performed extensive analyses on the top genes in this list as high confidence CREB targets. We found that this list is enriched for genes involved in cancer, and unexpectedly, highly enriched for histone genes. Furthermore, histone genes regulated by CREB were more likely to be specifically expressed in hematopoietic lineages. Decreased expression of specific histone genes was validated in K562, TF-1, and primary AML cells transduced with CREB shRNA. CONCLUSION: We have identified a high confidence list of CREB targets in K562 cells. These genes allow us to begin to understand the mechanisms by which CREB contributes to acute leukemia. We speculate that regulation of histone genes may play an important role by possibly altering the regulation of DNA replication during the cell cycle.

CREB is a critical regulator of normal hematopoiesis and leukemogenesis.

The cAMP-responsive element binding protein (CREB) is a 43-kDa nuclear transcription factor that regulates cell growth, memory, and glucose homeostasis. We showed previously that CREB is amplified in myeloid leukemia blasts and expressed at higher levels in leukemia stem cells from patients with myeloid leukemia. CREB transgenic mice develop myeloproliferative disease after 1 year, but not leukemia, suggesting that CREB contributes to but is not sufficient for leukemogenesis. Here, we show that CREB is most highly expressed in lineage negative hematopoietic stem cells (HSCs). To understand the role of CREB in hematopoietic progenitors and leukemia cells, we examined the effects of RNA interference (RNAi) to knock down CREB expression in vitro and in vivo. Transduction of primary HSCs or myeloid leukemia cells with lentiviral CREB shRNAs resulted in decreased proliferation of stem cells, cell- cycle abnormalities, and inhibition of CREB transcription. Mice that received transplants of bone marrow transduced with CREB shRNA had decreased committed progenitors compared with control mice. Mice injected with Ba/F3 cells expressing either Bcr-Abl wild-type or T315I mutation with CREB shRNA had delayed leukemic infiltration by bioluminescence imaging and prolonged median survival. Our results suggest that CREB is critical for normal myelopoiesis and leukemia cell proliferation.

Potential role of CREB as a prognostic marker in acute myeloid leukemia.

The cAMP response element binding protein (CREB) is a leucine zipper transcription factor that regulates genes responsible for cell proliferation, differentiation and survival. CREB is overexpressed in the bone marrow from most patients with acute leukemia. Overexpression of CREB occurs both at the protein and at the transcript levels and is associated with gene amplification in leukemic blast cells. Higher levels of CREB correlate with a less favorable prognosis in a small cohort of adult patients with acute myeloid leukemia. In one study, patients whose bone marrow over-expresses CREB had an increased risk of relapse and decreased event-free survival. Mice that overexpress CREB in myeloid cells develop a myeloproliferative/myelodysplastic syndrome. These findings suggest that CREB plays an important role in the pathogenesis of acute leukemia and is a potential biomarker of disease.

Role of cyclic AMP response element binding protein in human leukemias.

Acute myeloid leukemia (AML) in adults has a 20% 5-year disease-free survival despite treatment with aggressive cytotoxic chemotherapy. Previous work from our laboratory demonstrated that the majority of patients with acute lymphoid and myeloid leukemia overexpress CREB in the bone marrow. CREB overexpression is associated with poor initial outcome of clinical disease in AML patients. CREB is a transcription factor that functions in glucose homeostasis, growth-factor-dependent cell survival, and memory. Signaling by hematopoietic growth factors, such as GM-CSF, results in activation of CREB and up-regulation of CREB target genes. To study its role in hematopoiesis, we overexpressed CREB in leukemia cell lines and in mice. CREB overexpression resulted in increased survival and proliferation of myeloid cells and blast-transformation of bone marrow progenitor cells from transgenic mice expressing CREB in the myeloid lineage. CREB transgenic mice also develop myeloproliferative disease after 1 year. Thus, CREB acts as a protooncogene to regulate hematopoiesis and contributes to the leukemia phenotype. Our results suggest that CREB-dependent pathways may serve as targets for directed therapies in leukemia in the future.

Improved documentation of wound care with a structured encounter form in the pediatric emergency department.

OBJECTIVE: Accurate and complete documentation may enhance reimbursement and compliance with financial intermediary regulations, protect against litigation, and improve patient care. We measured the effect of introduction of a structured encounter form on the completeness of documentation of pediatric wound management in a teaching hospital. METHODS: The Children's Hospital Emergency Department introduced a structured encounter form for use in the documentation of wound care in place of the existing free-text dictation method. Attending physicians and trainees, all unaware of the study, had the option of using the form in place of free-text dictation for patients with lacerations requiring closure. We abstracted 100 consecutive free-text dictations from patients treated before the form's introduction. Following a 3-month run-in period, we abstracted 100 consecutive structured wound records. We compared the 2 chart types for completeness of documentation based on 20 predetermined criteria relevant to pediatric wound care. RESULTS: Overall completeness of documentation improved with structured forms (80% vs 68% for free text, P < .001), with significant improvements in 6 of 20 individual criteria. Trainees demonstrated improvement in documentation with the structured form, with the greatest improvements among senior-level residents. Documentation of the general physical examination worsened with structured charting. DISCUSSION: In an academic pediatric emergency department, the use of a structured complaint-specific form improved overall completeness of wound-care documentation. Structured encounter forms may provide for more standardized documentation for a variety of pediatric chief complaints, thereby facilitating communication and ultimately transition to template-driven systems in anticipation of an electronic medical record.

The role of CREB as a proto-oncogene in hematopoiesis and in acute myeloid leukemia.

CREB is a transcription factor that functions in glucose homeostasis, growth factor-dependent cell survival, and memory. In this study, we describe a role of CREB in human cancer. CREB overexpression is associated with increased risk of relapse and decreased event-free survival. CREB levels are elevated in blast cells from patients with acute myeloid leukemia. To understand the role of CREB in leukemogenesis, we studied the biological consequences of CREB overexpression in primary human leukemia cells, leukemia cell lines, and transgenic mice. Our results demonstrate that CREB promotes abnormal proliferation and survival of myeloid cells in vitro and in vivo through upregulation of specific target genes. Thus, we report that CREB is implicated in myeloid cell transformation.

Severe lactic acidosis in a 14-year-old female with metastatic undifferentiated carcinoma of unknown primary.

A 14-year-old girl was found to have a large, non-tender breast mass with anemia and thrombocytopenia. The diagnosis of an undifferentiated carcinoma of unknown primary was made after open breast biopsy of the mass with negative immunohistochemical studies for breast malignancies. Further evaluation showed extensive metastatic disease affecting the bone marrow, ribs, liver, and brain with magnetic resonance imaging evidence of carcinomatous meningitis. Despite 2 months of chemotherapy and intensive supportive care, the patient died of severe lactic acidosis and disseminated intravascular coagulation after exaggerated menstrual bleeding. The association of severe lactic acidosis and undifferentiated carcinoma of unknown primary in an adolescent has not been previously described.

The emerging role of RNA interference in the design of novel therapeutics in oncology.

RNA interference (RNAi) has emerged as the tool of choice for studying gene function. Dubbed the "breakthrough of the year" in 2002 by the journal Science, RNAi is a naturally occurring host defense mechanism that mediates the sequence-specific degradation of target mRNA transcripts and their protein products. The specificity of RNAi makes it an ideal tool for targeted therapeutics against unique fusion oncogene sequences. RNAi may also be effective against viral-mediated oncogenesis and has the potential to enhance tumor sensitivities to existing chemotherapy. The current interest in the success of RNAi-based therapies will depend on the delivery systems that protect the silencing apparatus from endogenous nucleases, sustain tissue-specific expression of the small-interfering RNAs, and prevent the activation of a destructive nonspecific host immune response.

RNA interference and human disease.

The completion of the human genome project has left researchers searching for an efficient method to study gene function in mammalian cells. RNA interference (RNAi) is an evolutionarily conserved post-transcriptional gene silencing (PTGS) mechanism mediated by double-stranded RNA (dsRNA). The dsRNA is processed into small duplex RNA molecules of approximately 21-22 nucleotides (nts) termed small interfering RNAs (siRNAs) by a RNase III enzyme called Dicer. Interaction of siRNAs with a multi-protein complex, termed the RNA-induced silencing complex (RISC), results in sequence specific association of the activated RISC complex with the cognate RNA transcript. This interaction leads to sequence-specific cleavage of the target transcript. Originally discovered in Caenorhabditis elegans, the study of RNAi in mammalian cells has blossomed in the last couple of years with the discovery that introduction of siRNA molecules directly into somatic mammalian cells circumvents the non-specific response vertebrate cells have against larger dsRNA molecules. Emerging as a powerful tool for reverse genetic analysis, RNAi is rapidly being applied to study the function of many genes associated with human disease, in particular those associated with oncogenesis and infectious disease. This review summarizes the mechanism of RNAi and provides an overview of its current applications in medicine.