Successful reduced-intensity conditioning hematopoietic stem cell transplantation for paroxysmal nocturnal hemoglobinuria with aplastic anemia in two children.

Paroxysmal nocturnal hemoglobinuria (PNH) is an extremely rare cause of bone marrow failure in children. We report two children who presented with pancytopenia, and were diagnosed with PNH with severe aplastic anemia. Both children underwent upfront, successful hematopoietic stem cell transplantation with reduced-intensity conditioning. One patient had a syngeneic donor, and one patient had a 10/10 matched unrelated donor. Neither patient developed graft versus host disease, infections, or recurrent PNH. Reduced-intensity conditioning hematopoietic stem cell transplantation is a reasonable therapy for PNH with marrow failure in children.

Patterns of Diagnosis and Misdiagnosis in Pediatric Cancer and Relationship to Survival.

BACKGROUND: Pediatric cancer is rare and its symptoms are often ambiguous. The aims of this study were to investigate the time needed to make a diagnosis, assess the frequency of misdiagnosis, and to determine whether these factors affected survival. METHODS: A review of records of 364 pediatric patients diagnosed with cancer at the University of Rochester Golisano Children's Hospital between 2004 and 2012 was conducted. Data were extracted on patient and health care system-related factors and clinical outcomes. RESULTS: The median time from symptom onset to diagnosis was shortest for leukemia (18.5 d) and longest for bone tumors (86.5 d). Tumor type was the only factor associated with time to diagnosis. In 52% of cases an incorrect nononcological diagnosis was initially made. Soft tissue sarcomas and brain tumors were misdiagnosed most often. Neither prolonged time to diagnosis nor initial misdiagnosis was associated with reduced survival. Tumor type and presence of metastatic disease at diagnosis were significantly associated with survival. CONCLUSIONS: There is significant variation in the time from symptom onset to diagnosis of pediatric cancers, and incorrect initial diagnostic impressions are common. Despite this there is no impact of prolonged time to diagnosis on survival.

Efficacy and manageable safety of tagraxofusp in blastic plasmacytoid dendritic cell neoplasm: a case series of pediatric and adolescent/young adult patients.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) predominantly occurs in adults ≥60 years old; 10-20% of cases are pediatric or adolescent/young adult (AYA) patients. Tagraxofusp (TAG, Elzonris®) is the only approved treatment for BPDCN; in the United States it is approved for patients aged ≥2 years. Data on treating pediatric and AYA BPDCN patients are limited. We present a case series of pediatric and AYA patients with BPDCN treated with TAG. Eight patients (five newly diagnosed; three relapsed/refractory [R/R]), aged 2-21 years, received 12 mcg/kg TAG. Seven patients were female; most had skin (n = 6) and/or bone marrow (n = 4) involvement. No new safety signals were identified. Grade 3 adverse events were headache (n = 1) and transaminitis (n = 2). Three patients with newly diagnosed BPDCN achieved complete response, one achieved partial response, and one had stable disease (SD). One patient with R/R BPDCN achieved a minor response; one had SD. Seven patients (88%) were bridged to stem cell transplant: 80% of newly diagnosed patients and 100% of R/R patients. Five patients remained alive at last follow-up. These cases highlight the efficacy and safety of TAG in pediatric and AYA patients for whom there is no other approved BPDCN therapy.

Clinical practice guideline for the use of antimicrobial agents in neutropenic patients with cancer: 2010 update by the infectious diseases society of america.

This document updates and expands the initial Infectious Diseases Society of America (IDSA) Fever and Neutropenia Guideline that was published in 1997 and first updated in 2002. It is intended as a guide for the use of antimicrobial agents in managing patients with cancer who experience chemotherapy-induced fever and neutropenia. Recent advances in antimicrobial drug development and technology, clinical trial results, and extensive clinical experience have informed the approaches and recommendations herein. Because the previous iteration of this guideline in 2002, we have a developed a clearer definition of which populations of patients with cancer may benefit most from antibiotic, antifungal, and antiviral prophylaxis. Furthermore, categorizing neutropenic patients as being at high risk or low risk for infection according to presenting signs and symptoms, underlying cancer, type of therapy, and medical comorbidities has become essential to the treatment algorithm. Risk stratification is a recommended starting point for managing patients with fever and neutropenia. In addition, earlier detection of invasive fungal infections has led to debate regarding optimal use of empirical or preemptive antifungal therapy, although algorithms are still evolving. What has not changed is the indication for immediate empirical antibiotic therapy. It remains true that all patients who present with fever and neutropenia should be treated swiftly and broadly with antibiotics to treat both gram-positive and gram-negative pathogens. Finally, we note that all Panel members are from institutions in the United States or Canada; thus, these guidelines were developed in the context of North American practices. Some recommendations may not be as applicable outside of North America, in areas where differences in available antibiotics, in the predominant pathogens, and/or in health care-associated economic conditions exist. Regardless of venue, clinical vigilance and immediate treatment are the universal keys to managing neutropenic patients with fever and/or infection.

Clinical practice guideline for the use of antimicrobial agents in neutropenic patients with cancer: 2010 Update by the Infectious Diseases Society of America.

This document updates and expands the initial Infectious Diseases Society of America (IDSA) Fever and Neutropenia Guideline that was published in 1997 and first updated in 2002. It is intended as a guide for the use of antimicrobial agents in managing patients with cancer who experience chemotherapy-induced fever and neutropenia. Recent advances in antimicrobial drug development and technology, clinical trial results, and extensive clinical experience have informed the approaches and recommendations herein. Because the previous iteration of this guideline in 2002, we have a developed a clearer definition of which populations of patients with cancer may benefit most from antibiotic, antifungal, and antiviral prophylaxis. Furthermore, categorizing neutropenic patients as being at high risk or low risk for infection according to presenting signs and symptoms, underlying cancer, type of therapy, and medical comorbidities has become essential to the treatment algorithm. Risk stratification is a recommended starting point for managing patients with fever and neutropenia. In addition, earlier detection of invasive fungal infections has led to debate regarding optimal use of empirical or preemptive antifungal therapy, although algorithms are still evolving. What has not changed is the indication for immediate empirical antibiotic therapy. It remains true that all patients who present with fever and neutropenia should be treated swiftly and broadly with antibiotics to treat both gram-positive and gram-negative pathogens. Finally, we note that all Panel members are from institutions in the United States or Canada; thus, these guidelines were developed in the context of North American practices. Some recommendations may not be as applicable outside of North America, in areas where differences in available antibiotics, in the predominant pathogens, and/or in health care-associated economic conditions exist. Regardless of venue, clinical vigilance and immediate treatment are the universal keys to managing neutropenic patients with fever and/or infection.

Evidence of B cell immune responses to acute lymphoblastic leukemia in murine allogeneic hematopoietic stem cell transplantation recipients treated with donor lymphocyte infusion and/or vaccination.

These experiments explored mechanisms of control of acute lymphoblastic leukemia (ALL) following allogeneic hematopoietic stem cell transplantation using a murine model of MHC-matched, minor histocompatibility antigen-mismatched transplantation. The central hypothesis examined was that addition of active vaccination against leukemia cells would substantially increase the effectiveness of allogeneic donor lymphocyte infusion (DLI) against ALL present in the host after transplantation. Although vaccination did increase the magnitude of type I T cell responses against leukemia cells associated with DLI, it did not lead to substantial improvement in long-term survival. Analysis of immunologic mechanisms of leukemia progression demonstrated that the failure of vaccination was not because of antigen loss in leukemia cells. However, analysis of survival provided surprising findings that, in addition to very modest type I T cell responses, a B cell response that produced antibodies that bind leukemia cells was found in long-term survivors. The risk of death from leukemia was significantly lower in recipients that had higher levels of such antibodies. These studies raise the hypothesis that stimulation of B cell responses after transplantation may provide a novel way to enhance allogeneic graft-versus-leukemia effects associated with transplantation.

Gene Expression and Survival of Acute Lymphoblastic Leukemia Cells After Allogeneic Transplant.

BACKGROUND/AIM: This study explored the mechanisms of the allogeneic graft versus leukemia effect in acute lymphoblastic leukemia (ALL) cells by examining whether they change gene expression in the post-transplant environment containing cytokines and the immunosuppressant cyclosporine, and if such changes affect ALL cell survival. MATERIALS AND METHODS: RNASeq was used to assess leukemia global gene expression and flow cytometry to measure ALL survival in the presence of T cells, NK cells, cytokines, and cyclosporine. RESULTS: A total of 4,805 genes were differentially expressed. Gene set enrichment analysis demonstrated up-regulation of biological processes related to cytokine responses, control of viral infection, and regulation of leukocyte function including proliferation. Down-regulated genes were related to mesenchymal tissue morphogenesis. ALL cells exposed to cytokines and cyclosporine retained susceptibility to T and NK cell killing, and also exhibited increased cell death without exposure to killer cells. CONCLUSION: A significant portion of the graft versus leukemia effect may be mediated by cytokines and cyclosporine.

Differential gene expression in acute lymphoblastic leukemia cells surviving allogeneic transplant.

The effectiveness of allogeneic graft-versus-leukemia (GVL) activity in control of acute lymphoblastic leukemia is generally regarded as poor. One possible factor is dynamic adaptation of the leukemia cell to the allogeneic environment. This work tested the hypothesis that the pattern of gene expression in acute lymphoblastic leukemia cells in an allogeneic environment would differ from that in a non-allogeneic environment. Expression microarray studies were performed in murine B lineage acute lymphoblastic leukemia cells recovered from mice that had undergone allogeneic MHC-matched but minor histocompatibility antigen mismatched transplants. A limited number of genes were found to be differentially expressed in ALL cells surviving in the allogeneic environment. Functional analysis demonstrated that genes related to immune processes, antigen presentation, ubiquitination and GTPase function were significantly enriched. Several genes with known immune activities potentially relevant to leukemia survival (Ly6a/Sca-1, TRAIL and H2-T23) were examined in independent validation experiments. Increased expression in vivo in allogeneic hosts was observed, and could be mimicked in vitro with soluble supernatants of mixed lymphocyte reactions or interferon-gamma. The changes in gene expression were reversible when the leukemia cells were removed from the allogeneic environment. These findings suggest that acute lymphoblastic leukemia cells respond to cytokines present after allogeneic transplantation and that these changes may reduce the effectiveness of GVL activity.

Risk factors for umbilical venous catheter-associated thrombosis in very low birth weight infants.

BACKGROUND: Thrombosis in neonates is a rare but serious occurrence, usually associated with central catheterization. The objective of this study was to investigate the risk factors associated with catheter related thrombosis in very low birth weight (VLBW) infants. PROCEDURE: The present retrospective study was performed using data from a randomized trial of duration of umbilical venous catheters (UVC) placement among infants <1,250 g birth weight. Twenty-two cases of UVC-associated thrombosis were identified in this sample. The remaining study sample (n = 188) served as the comparison group. Data on thrombosis, platelets, gestational age, birth weight, hematocrit, serum sodium, maternal preeclampsia, blood group, infant of diabetic mother (IDM) and demographic factors were collected using database and record review. RESULTS: Among the total subjects (n = 210), 112 (53%) were males and 126 (60%) were Caucasians, with mean gestational age of 27.7 +/- 2.1 weeks (standard deviation) and mean birth weight of 923 +/- 195 g. Bivariate analysis revealed significant association of thrombosis with hematocrit >55% in the first week (odds ratio [OR] 5.4; 95% confidence interval [CI] 2.0-14.6; P = 0.0003), being small for gestational age (SGA) (OR, 2.9; 95% CI, 1.2-7.4; P = 0.02) and maternal preeclampsia (OR, 3.97; 95% CI, 1.6-9.84; P = 0.0017). In multivariate logistic regression analysis, only hematocrit >55% was independently associated with thrombus (OR, 3.7; 95% CI 1.1-11.8; P = 0.03). CONCLUSIONS: This study demonstrates a significant, independent association between elevated hematocrit and development of UVC-associated thrombosis. Careful monitoring for catheter-associated thrombosis may be indicated in VLBW infants who have hematocrit >55% in the first week of life.

Support of acute lymphoblastic leukemia cells by nonmalignant bone marrow stromal cells.

The present report describes work examining the manner in which nonmalignant bone marrow stromal cells prevent acute lymphoblastic leukemia (ALL) cell death. The initial focus was on the role of stromal cell-derived C-X-C motif chemokine 12 (CXCL12). Interference with CXCL12 production by stroma or blockade of its interactions with ALL by plerixafor did increase ALL cell death and in sensitive ALLs there was synergistic effect with conventional chemotherapy drugs. However, in contrast to most reports, there was considerable heterogeneity regarding the effect between 7 unique primary ALLs, with several exhibiting no sensitivity to CXCL12 blockade. The diversity in effect was not explained by differences in the expression of ALL cell surface receptors for CXCL12. The modest and variable effects of interference with CXCL12 on ALL led to the assessment of gene expression profiles of stromal cells and ALL cells. Gene set enrichment analysis identified pathways associated with metabolism and redox reactions as potentially important in the stromal cell: leukemia cell interaction. Exploratory imaging studies demonstrated bidirectional transfer of intracellular calcien-labelled molecules and also bidirectional transfer of mitochondria between stromal cells and ALL cells, providing potential means of metabolic interdependence of stromal cells and ALL cells.

High-risk acute lymphoblastic leukemia cells with bcr-abl and INK4A/ARF mutations retain susceptibility to alloreactive T cells.

INK4A/ARF mutations are acquired in bcr/abl(+) lymphoid blast phase chronic myelogenous leukemia (CML) and bcr/abl(+) acute lymphoblastic leukemia (ALL). Donor lymphocyte infusion and graft-versus-leukemia (GVL) are generally ineffective in such ALLs, whereas GVL is highly active against bcr/abl(+) CML, which does not have a lesion in the INK4A/ARF locus. The mechanisms for the ineffectiveness of GVL are not fully known, and it is possible that intrinsic resistance of acute lymphoid leukemias to immune effectors associated with allogeneic GVL may contribute to ineffectiveness. This work tested the hypothesis that INK4A/ARF mutations that are associated with transformation of bcr/abl(+) CML to an ALL phenotype, and that are associated with increased resistance to apoptosis render ALL cells insensitive to allogeneic immune responses to minor histocompatibility antigens (mHA). Murine acute pre-B ALLs were induced by transfer of the human p210 bcr/abl gene into bone marrow of INK4A/ARF null mice. These ALL lines were then studied in a murine model of MHC-matched, mHA-mismatched allogeneic BMT. In vivo growth of these ALLs was inhibited in allogeneic transplants characterized by active allogeneic immune responses compared to their behavior in syngeneic transplants. In vitro ALLs with INK4A/ARF, p210 bcr/abl, or p190 bcr/abl mutations remained sensitive to anti-mHA cytolytic T cells. In addition, the ALLs were capable of inducing primary immune responses to mHAs in vivo. Thus, ALLs with INK4A/ARF or bcr/abl mutations are not intrinsically resistant to allogeneic T cell responses, suggesting that active immunotherapies against mHA have the potential to control such acute lymphoblastic leukemias.

Down-regulation of antihost alloreactivity after bone marrow transplant permits relapse of hematological malignancy.

Relapse of leukemia remains a common event after allogeneic bone marrow transplantation, despite potential donor antihost alloreactivity present in most transplants. This work examined posttransplant relapse of the DBA/2 P815 mastocytoma in a murine model of MHC-matched, minor histocompatibility antigen (mHAg)-mismatched bone marrow transplantation (BALB/c donors into DBA/2 recipients). Antihost alloreactivity was associated with reduction of posttransplant tumor burden and prolongation of survival, but posttransplant relapse commonly occurred. No evidence of acquired resistance to immune control was found in 12 relapse reisolates. Relapse tumors remained sensitive to donor antihost CTLs in vitro, suggesting continued expression of mHAgs. Reisolates also continued to express Fas. However, loss of posttransplant alloreactivity was observed at 3 weeks. This was temporally associated with the time of relapse. Antihost alloreactivity could be reactivated in stable graft-versus-host disease-free recipients by immunization with host cells. The results of this study suggest that one mechanism for relapse after bone marrow transplant is acquired tolerance of allogeneic minor histocompatibility antigens and that posttransplant immunotherapy directed against mHAgs may induce antitumor activity.

Original Research: Acute chest syndrome in sickle cell disease: Effect of genotype and asthma.

Sickle cell disease is a severe hemoglobinopathy caused by mutations in the beta globin genes. The disorder has protean manifestations and leads to severe morbidity and early mortality. Acute chest syndrome (ACS) is a common complication and in the USA is the leading cause of death in patients with sickle cell disease. Care of patients with sickle cell disease is complex and typically involves both primary care physicians and hematology subspecialists. The purpose of this study was first to attempt to validate in a pediatric sickle cell patient cohort associations between ACS and sickle cell disease genotype and between ACS and asthma as a comorbidity. The second purpose of the study was to study in a typical community the frequency with which asthma associated with ACS was addressed in terms of electronic medical record integration, pulmonary subspecialty consultation for management of asthma, and completion of pulmonary function testing (PFTs). A retrospective study of the electronic medical record of a children's hospital that provides most of the medical care for children in a portion of western New York state was performed. We found that ACS was more common in the sickle cell disease genotypes SS and S/beta-thalassemia-null, and that ACS was more frequent in patients treated for asthma. We also found that despite the use of a comprehensive electronic medical record, there was poor documentation of ACS and asthma episodes in the problem lists of patients with sickle cell disease, and that most patients with sickle cell disease with ACS or asthma failed to receive formal consultation services from pediatric pulmonary subspecialists.

Cellular tumor vaccines administered after T cell-depleted allogeneic bone marrow transplantation induce effective anti-tumor immune responses.

In allogeneic hematopoietic stem cell (HSC) transplantation, graft vs. tumor (GVT) activity contributes to the cancer cure. It is closely associated with graft vs. host disease (GVHD), an immune response initiated by transplanted donor T-cell responses against host minor histocompatibility antigens (mHAgs). GVHD is prevented by T-cell depletion of the donor graft, but T-cell depletion also abrogates curative GVT. We wished to test the hypothesis that cellular tumor vaccines administered after T cell-depleted HSC transplant can induce significant GVT effects, despite the absence of transplanted mature donor T cells. In this investigation, a murine model of major histocompatibility complex (MHC)-matched, mHAg-mismatched allogeneic HSC transplant was studied. T cell-depleted or normal T cell-containing grafts were given to myeloablated recipients. Following reconstitution the recipients were vaccinated with tumor vaccines. GVT responses were measured in vitro by T-cell function assays and flow cytometry, and in vivo by tumor burden or survival. Post-transplant tumor vaccines induced effective anti-tumor responses in recipients of T cell-depleted transplants, producing cytolytic and cytokine responses, reduced tumor burden and prolonged survival. Recipients of T cell-depleted transplants that still have significant thymic function may be suitable subjects for post-transplant vaccine therapy.

Ciprofloxacin in treatment of fever and neutropenia in pediatric cancer patients.

This article reviews clinical trials of outpatient management of fever and neutropenia in pediatric cancer patients. The syndrome of fever and neutropenia is discussed, and strategies of identifying patients at low risk for complex or fatal infections are described. A number of clinical trials in a wide range of clinical settings and countries have demonstrated that low risk pediatric cancer patients with fever and neutropenia can be prospectively identified and safely treated as outpatients. In addition outpatient management has been shown to be less costly than conventional intravenous therapy in hospitalized patients. Oral fluoroquinolones, including ciprofloxacin, have been used as a component of therapy in several trials because of their ease of administration and their activity against the majority of pathogenic bacteria causing illness in this group. The article also discusses the role of antibiotic prophylaxis of fever and neutropenia in certain high risk settings, such as hematopoietic stem cell transplantation. In selected high risk patients, prophylactic use of limited spectrum fluoroquinolones such as ciprofloxacin may reduce the incidence of Gram-negative bacteremias. Use of fluoroquinolone therapy as prophylaxis, however, is controversial because of concerns about an emergence of resistant organisms. Prudent use of fluoroquinolones as therapy and prophylaxis is essential to prolonging the benefits of this class of compounds.

Suicide gene therapy using E. coli beta-galactosidase.

PURPOSE: Suicide gene therapy offers the potential to increase the selective toxicity of antitumor agents by intratumoral expression of exogenous enzymes that convert nontoxic prodrugs to toxic products. The use of herpes simplex virus thymidine kinase with ganciclovir, and E. coli cytosine deaminase with 5-fluorocytosine are well-known examples of this approach. The purpose of this study was to investigate a novel suicide gene therapy using E. coli beta-galactosidase (beta-gal) as the prodrug-activating enzyme. Advantages of this approach include: (1) the ability to use prodrugs that are cleaved by beta-gal to agents that are known to possess activity against human solid tumors, and (2) the extensive experience gained with targeting beta-gal to specific tumors in experimental animals and in humans. METHODS: Two different structural types of anthracycline prodrugs, N-[4"-(beta- D-galactopyranosyl)-3"-nitrobenzyloxycarbonyl]daunomycin (Daun02) and N-[(4" R,S)-4"-ethoxy-4"-(1"'- O-beta- D-galactopyranosyl)butyl]daunorubicin (gal-DNC4) were investigated. The prodrugs were evaluated as substrates for beta-gal. Cytotoxicity studies of Daun02 were conducted against a murine tumor (Panc02), two human breast tumors (MCF-7 and T47D), and three human prostate tumors (PC3, DU145 and LNCAP) that had been transduced to express beta-gal. Antitumor studies of Daun02 were conducted against mouse tumor Panc02 xenografts implanted subcutaneously. RESULTS: Daun02 was a good substrate for beta-gal. By comparison, gal-DCN4 was a poor substrate. Except for PC3, the beta-gal-transduced tumors showed 3- to 60-fold increased sensitivity to Daun02 compared with mock-transduced control cells. Daunomycin was formed from Daun02 in tissue culture medium containing beta-gal-transduced cell lines but was not observed in the medium from mock-transduced controls. In vivo therapeutic studies of Daun02 against the Panc02 tumor in athymic mice showed no significant inhibition of tumor growth. Pharmacokinetic studies showed limited distribution of the prodrug beyond the vascular space. CONCLUSIONS: E. coli beta-gal may be useful as a prodrug-activating enzyme in suicide gene therapy and has the potential to increase the selective toxicity of conventional antitumor agents. Although this approach worked well against tumor cells in vitro, it was not effective against a xenograft model in vivo, apparently because of poor drug-tissue distribution.