Long-Term Outcomes after Treatment with Clofarabine ± Fludarabine with Once-Daily Intravenous Busulfan as Pretransplant Conditioning Therapy for Advanced Myeloid Leukemia and Myelodysplastic Syndrome.

Pretransplant conditioning regimens critically determine outcomes in the setting of allogeneic stem cell transplantation (allo-SCT). The use of nucleoside analogs such as fludarabine (Flu) in combination with i.v. busulfan (Bu) has been shown to be highly effective as a pretransplant conditioning regimen in acute myeloid leukemia (AML), chronic myeloid leukemia (CML), and myelodysplastic syndrome (MDS). Because leukemia relapse remains the leading cause of death after allo-SCT, we studied whether clofarabine (Clo), a nucleoside analog with potent antileukemia activity, can be used to complement Flu. In a preliminary report, we previously showed the safety and efficacy of Clo ± Flu with i.v. Bu in 51 patients with high-risk AML, CML, and MDS. The study has now been completed, and we present long-term follow-up data on the entire 70-patient population, which included 49 (70%), 8 (11%), and 13 (19%) patients with AML, MDS, and CML, respectively. Thirteen patients (19%) were in complete remission, and 41 patients (59%) received matched unrelated donor grafts. Engraftment was achieved in all patients. Sixty-three patients (90%) achieved complete remission. There were no deaths reported at day +30, and the 100-day nonrelapse mortality rate was 4% (n = 3). Thirty-one percent of patients (n = 22) developed grades II to IV acute graft-versus-host disease, and the median overall survival and progression-free survival times were 2.4 years and .9 years, respectively. Our results confirm the safety and overall and progression-free survival advantage of the arms with higher Clo doses and lower Flu doses, which was most prominent in the AML/MDS group.

Autologous bone marrow mononuclear cells reduce therapeutic intensity for severe traumatic brain injury in children.

OBJECTIVES: The devastating effect of traumatic brain injury is exacerbated by an acute secondary neuroinflammatory response, clinically manifest as elevated intracranial pressure due to cerebral edema. The treatment effect of cell-based therapies in the acute post-traumatic brain injury period has not been clinically studied although preclinical data demonstrate that bone marrow-derived mononuclear cell infusion down-regulates the inflammatory response. Our study evaluates whether pediatric traumatic brain injury patients receiving IV autologous bone marrow-derived mononuclear cells within 48 hours of injury experienced a reduction in therapeutic intensity directed toward managing elevated intracranial pressure relative to matched controls. DESIGN: The study was a retrospective cohort design comparing pediatric patients in a phase I clinical trial treated with IV autologous bone marrow-derived mononuclear cells (n = 10) to a control group of age- and severity-matched children (n = 19). SETTING: The study setting was at Children's Memorial Hermann Hospital, an American College of Surgeons Level 1 Pediatric Trauma Center and teaching hospital for the University of Texas Health Science Center at Houston from 2000 to 2008. PATIENTS: Study patients were 5-14 years with postresuscitation Glasgow Coma Scale scores of 5-8. INTERVENTIONS: The treatment group received 6 million autologous bone marrow-derived mononuclear cells/kg body weight IV within 48 hours of injury. The control group was treated in an identical fashion, per standard of care, guided by our traumatic brain injury management protocol, derived from American Association of Neurological Surgeons guidelines. MEASUREMENTS AND MAIN RESULTS: The primary measure was the Pediatric Intensity Level of Therapy scale used to quantify treatment of elevated intracranial pressure. Secondary measures included the Pediatric Logistic Organ Dysfunction score and days of intracranial pressure monitoring as a surrogate for length of neurointensive care. A repeated-measure mixed model with marginal linear predictions identified a significant reduction in the Pediatric Intensity Level of Therapy score beginning at 24 hours posttreatment through week 1 (p < 0.05). This divergence was also reflected in the Pediatric Logistic Organ Dysfunction score following the first week. The duration of intracranial pressure monitoring was 8.2 ± 1.3 days in the treated group and 15.6 ± 3.5 days (p = 0.03) in the time-matched control group. CONCLUSIONS: IV autologous bone marrow-derived mononuclear cell therapy is associated with lower treatment intensity required to manage intracranial pressure, associated severity of organ injury, and duration of neurointensive care following severe traumatic brain injury. This may corroborate preclinical data that autologous bone marrow-derived mononuclear cell therapy attenuates the effects of inflammation in the early post-traumatic brain injury period.

Clofarabine ± fludarabine with once daily i.v. busulfan as pretransplant conditioning therapy for advanced myeloid leukemia and MDS.

Although a combination of i.v. busulfan (Bu) and fludarabine (Flu) is a safe, reduced-toxicity conditioning program for acute myelogenous leukemia/myelodysplastic syndromes (AML/MDS), recurrent leukemia posttransplantation remains a problem. To enhance the conditioning regimen's antileukemic effect, we decided to supplant Flu with clofarabine (Clo), and assayed the interactions of these nucleoside analogs alone and in combination with Bu in Bu-resistant human cell lines in vitro. We found pronounced synergy between each nucleoside and the alkylator but even more enhanced cytotoxic synergy when the nucleoside analogs were combined prior to exposing the cells to Bu. We then designed a 4-arm clinical trial in patients with myeloid leukemia undergoing allogeneic stem cell transplantation (allo-SCT). Patients were adaptively randomized as follows: Arm I-Clo:Flu 10:30 mg/m(2), Arm II-20:20 mg/m(2), Arm III-30:10 mg/m(2), and Arm IV-single-agent Clo at 40 mg/m(2). The nucleoside analog(s) were/was infused over 1 hour once daily for 4 days, followed on each day by Bu, infused over 3 hours to a pharmacokinetically targeted daily area under the curve (AUC) of 6000 µMol-min ± 10%. Fifty-one patients have been enrolled with a minimum follow-up exceeding 100 days. There were 32 males and 19 females, with a median age of 45 years (range: 6-59). Nine patients had chronic myeloid leukemia (CML) (BC: 2, second AP: 3, and tyrosine-kinase inhibitor refractory first chronic phase [CP]: 4). Forty-two patients had AML: 14 were induction failures, 8 in first chemotherapy-refractory relapse, 7 in untreated relapse, 3 in second or subsequent relapse, 4 were in second complete remission (CR), and 3 in second CR without platelet recovery (CRp), 2 were in high-risk CR1. Finally, 1 patient was in first CRp. Graft-versus-host disease (GVHD) prophylaxis was tacrolimus and mini-methorexate (MTX), and those who had an unrelated or 1 antigen-mismatched donor received low-dose rabbit-ATG (Thymoglobulin™). All patients engrafted. Forty-one patients had active leukemia at the time of transplant, and 35 achieved CR (85%). Twenty of the 42 AML patients and 5 of 9 CML patients are alive with a projected median overall survival (OS) of 23 months. Marrow and blood (T cell) chimerism studies at day +100 revealed that both in the lower-dose Clo groups (groups 1+2) and the higher-dose Clo groups (groups 3+4), the patients had a median of 100% donor (T cell)-derived DNA. There has been no secondary graft failure. In the first 100 days, 1 patient died of pneumonia, and 1 of liver GVHD. We conclude that (1) Clo ± Flu with i.v. Bu as pretransplant conditioning is safe in high-risk myeloid leukemia patients; (2) clofarabine is sufficiently immunosuppressive to support allo-SCT in myeloid leukemia; and (3) the median OS of 23 months in this high-risk patient population is encouraging. Additional studies to evaluate the antileukemic efficacy of Clo ± Flu with i.v. Bu as pretransplant conditioning therapy are warranted.

Corruption of the Fas pathway delays the pulmonary clearance of murine osteosarcoma cells, enhances their metastatic potential, and reduces the effect of aerosol gemcitabine.

PURPOSE: Pulmonary metastases continue to be a significant problem in osteosarcoma. Apoptosis dysfunction is known to influence tumor development. Fas (CD95, APO-1)/FasL is one of the most extensively studied apoptotic pathways. Because FasL is constitutively expressed in the lung, cells that express Fas should be eliminated by lung endothelium. Cells with low or no cell surface Fas expression may be able to evade this innate defense mechanism. The purpose of these studies was to evaluate Fas expression in osteosarcoma lung metastases and the effect of gemcitabine on Fas expression and tumor growth. EXPERIMENTAL DESIGN AND RESULTS: Using the K7M2 murine osteosarcoma model, Fas expression was quantified using immunohistochemistry. High levels of Fas were present in primary tumors, but no Fas expression was present in actively growing lung metastases. Blocking the Fas pathway using Fas-associated death domain dominant-negative delayed tumor cell clearance from the lung and increased metastatic potential. Treatment of mice with aerosol gemcitabine resulted in increased Fas expression and subsequent tumor regression. CONCLUSIONS: We conclude that corruption of the Fas pathway is critical to the ability of osteosarcoma cells to grow in the lung. Agents such as gemcitabine that up-regulate cell surface Fas expression may therefore be effective in treating osteosarcoma lung metastases. These data also suggest that an additional mechanism by which gemcitabine induces regression of osteosarcoma lung metastases is mediated by enhancing the sensitivity of the tumor cells to the constitutive FasL in the lung.

Cell therapies for traumatic brain injury.

Preliminary discoveries of the efficacy of cell therapy are currently being translated to clinical trials. Whereas a significant amount of work has been focused on cell therapy applications for a wide array of diseases, including cardiac disease, bone disease, hepatic disease, and cancer, there continues to be extraordinary anticipation that stem cells will advance the current therapeutic regimen for acute neurological disease. Traumatic brain injury is a devastating event for which current therapies are limited. In this report the authors discuss the current status of using adult stem cells to treat traumatic brain injury, including the basic cell types and potential mechanisms of action, preclinical data, and the initiation of clinical trials.

Expression of human glutathione S-transferase P1 mediates the chemosensitivity of osteosarcoma cells.

Chemoresistance is a major reason that patients with osteosarcoma fail to achieve a lasting chemotherapy response, and it contributes to disease relapse, progression, and death. Human glutathione S-transferase P1 (GSTP1), a phase II detoxification enzyme, contributes to chemoresistance in many cancers. However, the role of GSTP1 in osteosarcoma chemoresistance is ill defined. We hypothesized that GSTP1 has cytoprotective effects in human osteosarcoma. To assess this possibility, we used GSTP1 cDNA transfection or RNA interference to overexpress or suppress GSTP1 in osteosarcoma cells, and assessed the cytotoxic effect of chemotherapeutic agents on these cells. Our results showed that GSTP1 expression was up-regulated in osteosarcoma cells when they were treated with doxorubicin or cisplatin. GSTP1 overexpression in SAOS-2 osteosarcoma cells caused the cells to be more resistant to doxorubicin and cisplatin. In contrast, GSTP1 suppression in HOS cells caused more apoptosis and extensive DNA damage in response to doxorubicin and cisplatin. The cytotoxicity assay also showed that GSTP1 suppression caused a 2.5-fold increase in cell growth inhibition resulting from doxorubicin and cisplatin treatments [the IC(50)s are approximately 0.16 micromol/L (doxorubicin) and 1.8 micromol/L (cisplatin) for parental HOS versus 0.06 micromol/L (doxorubicin) and 0.75 micromol/L (cisplatin) for GSTP1-silenced HOS]. Moreover, GSTP1 suppression decreased the activation of extracellular signal-regulated kinase 1/2, which is induced by cisplatin and doxorubicin. Taken together, these findings show that GSTP1 contributes to doxorubicin and cisplatin resistance in osteosarcoma, which may be mediated in part by the activation of extracellular signal-regulated kinase 1/2. Targeting of GSTP1 combined with chemotherapy may have synergistic therapeutic effects on osteosarcoma.

Infections in 100 cord blood transplantations: spectrum of early and late posttransplant infections in adult and pediatric patients 1996-2005.

Cord blood-derived stem cells are successfully used in the treatment of cancer and congenital disorders in children. This alternative source of stem cells is also explored for adult cancer patients with limited donor options. However, delayed engraftment, prolonged neutropenia, secondary graft loss, and graft-versus-host disease (GVHD) in recipients of cord blood transplantation (CBT) make opportunistic infections a serious concern. We evaluated the spectrum of infections in adults and children undergoing CBT at our National Cancer Institute-designated comprehensive cancer center. The infection incidence rate ratio (total infection episodes/days at risk [survival after CBT] x 100) was 2.4 times higher in 35 adult patients than in 62 children, especially in adults with neutropenia (3 x higher) and GVHD (1.9 x higher). Ninety-two percent of fungal infection episodes occurred within 100 days after transplantation; half of these infections occurred in the first 30 days after CBT. Most bacterial infections (80%) were also diagnosed in the first 100 days, whereas late (>100 d) post-CBT cytomegalovirus and varicella zoster virus infections occurred only in children with chronic GVHD. Multivariate analysis showed that resolution of lymphocytopenia (> or =1000 cells/microL) (hazard ratio [HR] 0.71; p < 0.0001) and successful engraftment (HR 0.20; p < 0.0001) were associated with a low risk of serious infection. Children (HR 0.36; p < 0.0002) with sustained engraftment (HR 0.39; p < 0.004) and those with cancer in remission (HR 0.47; p < 0.007) were less likely to die from infection. More effective measures for surveillance and prevention of late cytomegalovirus and varicella zoster virus infections in children with CBT and chronic GVHD are needed.

Interleukin-12 up-regulates Fas expression in human osteosarcoma and Ewing's sarcoma cells by enhancing its promoter activity.

Interleukin-12 (IL-12) has shown significant antitumor activity in several preclinical animal tumor models. Our previous studies showed that IL-12 inhibited tumor growth in human osteosarcoma and Ewing's sarcoma animal model. Decreased Fas expression in osteosarcoma increased the lung metastatic potential. In this study, we further examined the mechanism of IL-12 antitumor activity and showed that IL-12 significantly increased Fas expression in both human osteosarcoma cells LM7 and Ewing's sarcoma cells TC71. Up-regulation of Fas expression increased their sensitivity to Fas-induced cell apoptosis. Constructs of the Fas promoter linked to a luciferase reporter gene were used to determine the promoter activity. IL-12 increased Fas promoter activity 4.2- and 4.9-fold in TC71 and LM7 cells, respectively. Time course studies have shown that recombinant IL-12 stimulated Fas promoter activity at 2 hours, reached the peak level at 4 hours, and then declined at 24 hours. To investigate whether IL-12 specifically enhanced Fas promoter activity, we determined whether another gene (E1A) was able to stimulate Fas promoter activity. We also evaluated effect of IL-12 on the topoisomerase IIalpha promoter. The results indicated that E1A but not IL-12 stimulated topoisomerase IIalpha promoter activity. E1A failed to increase Fas promoter activity. We also found that kappaB-Sp1 element at position -295 to -286 in Fas promoter was essential for IL-12-induced activation, and nuclear factor-kappaB transcription factor was activated after IL-12 treatment in TC71 cells. These results indicate that IL-12 up-regulates Fas expression in human osteosarcoma and Ewing's sarcoma by enhancing Fas promoter activity. Understanding this mechanism may lead to new therapeutic approaches for the treatment of sarcoma involving the use of IL-12.

Aerosol gene therapy with PEI: IL-12 eradicates osteosarcoma lung metastases.

PURPOSE: We determined whether polyethylenimine (PEI), a polycationic DNA carrier, can be used to deliver the interleukin (IL) 12 gene by aerosol to treat established osteosarcoma (OS) lung metastases in a nude mouse model. EXPERIMENTAL DESIGN: Tumor response was assessed using our OS lung metastases model. Treatment with aerosolized PEI containing the murine IL-12 gene (PEI:IL-12; 600 microl PEI and 2 mg IL-12) was given twice weekly for 5-6 weeks. RESULTS: Aerosol therapy for 2 weeks resulted in high expression of both the p35 and p40 subunits of IL-12 in the lungs but not in the livers of mice. Peak IL-12 mRNA expression was seen 24 h after a single aerosol PEI:IL-12 treatment. This expression gradually decreased with continued detection for up to 7 days. IL-12 protein was not detectable in plasma even after 6 weeks of aerosol therapy. The number of lung metastases in mice treated with aerosol PEI:IL-12 was decreased significantly (median, 0; range, 0-33) compared with mice that received PEI alone (median, 37.5; range, 11-125; P = 0.002). Nodule size was also significantly smaller in the aerosol PEI:IL-12 group with 87% of the nodules measuring 1 mm. Weekly aerosol PEI:IL-12 therapy was as effective as twice weekly therapy. CONCLUSIONS: Aerosol therapy resulted in selective gene expression and protein production in the tumor area. Aerosol PEI:IL-12 may avoid the systemic toxicities described previously in patients treated with i.v. IL-12. Because OS metastasizes almost exclusively to the lung, aerosol PEI:IL-12 therapy may provide a therapeutic option, which may be especially valuable.

Increased Fas expression reduces the metastatic potential of human osteosarcoma cells.

PURPOSE: The process of metastasis requires the single tumor cell that seeds the metastatic clone to complete a complex series of steps. Identifying factors responsible for these steps is essential in developing and improving targeted therapy for metastasis. Resistance to receptor-mediated cell death, such as the Fas/Fas ligand pathway, is one mechanism commonly exploited by metastatic cell populations. EXPERIMENTAL DESIGN AND RESULTS: LM7, a subline of the SAOS human osteosarcoma cell line with low Fas expression, was selected for its high metastatic potential in an experimental nude mouse model. When transfected with the full-length Fas gene (LM7-Fas), these cells expressed higher levels of Fas than the parental LM7 cells or LM7-neo control-transfected cells. These cells were also more sensitive to Fas-induced cell death than controls. When injected intravenously into nude mice, the LM7-Fas cell line produced a significantly lower incidence of tumor nodules than control cell lines. Lung weight and tumor nodule size were also decreased in those mice injected with LM7-Fas. Levels of Fas were quantified in osteosarcoma lung nodules from 17 patients. Eight samples were Fas negative, whereas the remaining 9 were only weakly positive compared with normal human liver (positive control). CONCLUSIONS: Our results demonstrate that altering Fas expression can impact the metastatic potential of osteosarcoma cells. We conclude that the increase of Fas on the surface of the LM7 osteosarcoma cells increased their sensitivity to Fas-induced cell death in the microenvironment of the lung, where Fas ligand is constitutively expressed. Thus, loss of Fas expression is one mechanism by which osteosarcoma cells may evade host resistance mechanisms in the lung, increasing metastatic potential. Fas may therefore be a new therapeutic target for osteosarcoma.

Exploring the Cancer Experiences of Young Adults in the Context of Stem Cell Transplantation.

BACKGROUND: Cancer is the leading cause of nonaccidental morbidity and mortality among young adults (YAs) in the United States. Stem cell transplantation (SCT), a treatment modality for a variety of YA malignancies, often requires prolonged hospitalization and immune-compromising treatment regimens. Stem cell transplantation may isolate YAs physically and emotionally, contributing to uncertainty about treatment processes, outcomes, and long-term sequelae. Studies in this population suggest that uncertainty can contribute to difficulty accomplishing basic developmental tasks. Few studies have examined the experiences of YAs in active cancer treatment, particularly those undergoing SCT. OBJECTIVES: This study explored the cancer experiences of YAs aged 18 to 25 years leading up to SCT and explored how YAs construct issues of uncertainty related to the transplantation experience. METHODS: Interviews with 14 YAs conducted within 24 hours of admission to undergo SCT were analyzed using thematic analysis from a medical ethnographic perspective. RESULTS: Themes emerged within 2 domains: relational and psychoemotional. The relational theme of "altered relationships" included the subthemes of "moving from" and "moving toward." The psychoemotional theme of the "power of perspective" included the subthemes of "optimism," "acknowledgment of death," "informational empowerment," and "developing a new outlook." CONCLUSIONS: Our findings offer new insights into the YA experience in the context of active cancer treatment, specifically how the cancer experience impacts relationships and how this experience is influenced by YAs' perspectives. IMPLICATIONS FOR PRACTICE: This study provides a foundation for addressing the psychosocial needs of YAs hospitalized for SCT, paying particular attention to the development of specific interventions.

Eradication of osteosarcoma lung metastases following intranasal interleukin-12 gene therapy using a nonviral polyethylenimine vector.

The use of adenoviral vectors for therapeutic delivery of genes via pulmonary application poses several problems in terms of immune responses. The purpose of this study was to determine whether polyethylenimine (PEI), a polycationic DNA carrier, can be used to deliver the IL-12 gene into the lungs of mice having microscopic osteosarcoma (OS) lung metastases. Incubation of SAOS-LM6 cells in vitro with PEI containing the murine IL-12 (mIL-12) gene (PEI:IL-12) resulted in expression of both the p35 and p40 subunits of IL-12 mRNA and production of mIL-12 protein. Using our newly developed OS nude mouse model, we demonstrated that treatment of mice using intranasal PEI:IL-12 resulted in significant IL-12 mRNA expression in the lung but not the liver. Furthermore, plasma IL-12 was undetectable after up to 4 weeks of intranasal PEI:IL-12 therapy given twice weekly. No IL-12 expression was seen following intranasal PEI therapy alone. The number of lung metastases in animals that received intranasal PEI:IL-12 twice weekly for 4 weeks starting 6 weeks after tumor inoculation was significantly decreased (median, 11; range, 0-47) compared with those that received PEI alone (median, 89; range, 2 to >200; P=.012). Also, the size of the nodules was significantly smaller in the PEI:IL-12-treated animals, with 90% measuring < or =0.5 mm in diameter compared with 56% in the PEI-alone group. Animals that received PEI alone also had numerous large nodules (3-6 mm) throughout the lungs. Intranasal therapy is a noninvasive way to administer agents and has the advantage of targeting the pulmonary region, resulting in higher concentrations in the tumor area. Additionally, delivery of IL-12 to the lung via the airway using PEI may avoid systemic toxicity. Because OS metastasizes almost exclusively to the lung, this may be a novel approach to the treatment of pulmonary OS metastases.

Fas expression inversely correlates with metastatic potential in osteosarcoma cells.

A complex series of steps must take place to allow for a single cell to metastasize. Identifying factors responsible for these steps is essential in developing targeted therapy. We developed series of osteosarcoma cell lines with differing metastatic potentials. We used them to investigate mechanisms of metastasis and possible therapeutic targets for osteosarcoma metastasis to the lung in a nude mouse model. No correlation was found between epidermal growth factor receptor (EGFR), insulin-like growth factor receptor inhibitor (IGF-I-R), gelatinase, p53, metalloproteinase 9 (MMP 9), platelet derived growth factor receptor (PDGF-R), vascular endothelial growth factor (VEGF) and c-met expression and metastatic potential as measured by Northern analysis. By contrast, Fas expression inversely correlated with metastatic potential, and manipulation of Fas expression altered the metastatic phenotype of the cell. Our data indicate that fas gene expression may offer a new therapeutic target for the treatment of metastatic osteosarcoma in the lung.

Fludarabine, melphalan, thiotepa and anti-thymocyte globulin conditioning for unrelated cord blood transplant.

Unrelated cord blood transplant (CBT) is an alternative treatment option for patients who lack a matched donor. However, the optimal type and intensity of the preparative regimen remains unclear. We evaluated the toxicity and outcomes of a conditioning regimen consisting of melphalan 140 mg/m(2) (day - 8), thiotepa 10 mg/kg (day - 7), fludarabine 160 mg/m(2) over 4 days (days - 6 to - 3) and rabbit antithymocyte globulin (ATG) 1.25 mg/kg (day - 4) and 1.75 mg/kg (day - 3) (FMT). Forty-seven patients with advanced hematologic malignancies with a median age of 23 years (30 adults and 17 children) were treated. Sixty percent of patients were in remission at transplant. Ninety-one percent of the patients engrafted neutrophils after a median of 22 days, and all but one of the patients achieving donor engraftment had hematopoietic recovery with 100% cord blood-derived cells. Grade 3 gastrointestinal toxicity was the major non-hematopoietic toxicity, occurring in 32% of patients. Cumulative incidence of day-100 grade II-IV acute graft-versus-host disease (aGVHD) and chronic graft-versus-host disease (cGVHD) was 53% and 34%, respectively, and non-relapse mortality at day 100 and 2 years was 11% and 40%. Two-year disease-free and overall survival rates were 31% and 44%, respectively. These results suggest that FMT is a feasible conditioning regimen for patients undergoing CBT.

Autologous bone marrow mononuclear cell therapy for severe traumatic brain injury in children.

BACKGROUND: Severe traumatic brain injury (TBI) in children is associated with substantial long-term morbidity and mortality. Currently, there are no successful neuroprotective/neuroreparative treatments for TBI. Numerous preclinical studies suggest that bone marrow-derived mononuclear cells (BMMNCs), their derivative cells (marrow stromal cells), or similar cells (umbilical cord blood cells) offer neuroprotection. OBJECTIVE: To determine whether autologous BMMNCs are a safe treatment for severe TBI in children. METHODS: Ten children aged 5 to 14 years with a postresuscitation Glasgow Coma Scale of 5 to 8 were treated with 6×10 autologous BMMNCs/kg body weight delivered intravenously within 48 hours after TBI. To determine the safety of the procedure, systemic and cerebral hemodynamics were monitored during bone marrow harvest; infusion-related toxicity was determined by pediatric logistic organ dysfunction (PELOD) scores, hepatic enzymes, Murray lung injury scores, and renal function. Conventional magnetic resonance imaging (cMRI) data were obtained at 1 and 6 months postinjury, as were neuropsychological and functional outcome measures. RESULTS: All patients survived. There were no episodes of harvest-related depression of systemic or cerebral hemodynamics. There was no detectable infusion-related toxicity as determined by PELOD score, hepatic enzymes, Murray lung injury scores, or renal function. cMRI imaging comparing gray matter, white matter, and CSF volumes showed no reduction from 1 to 6 months postinjury. Dichotomized Glasgow Outcome Score at 6 months showed 70% with good outcomes and 30% with moderate to severe disability. CONCLUSION: Bone marrow harvest and intravenous mononuclear cell infusion as treatment for severe TBI in children is logistically feasible and safe.

Production of VEGF165 by Ewing's sarcoma cells induces vasculogenesis and the incorporation of CD34+ stem cells into the expanding tumor vasculature.

The Ewing's sarcoma cell line TC71 overexpresses vascular endothelial growth factor isoform 165 (VEGF165), a potent proangiogenic molecule that induces endothelial cell proliferation, migration, and chemotaxis. CD34+ bone marrow stem cells can differentiate into endothelial and hematopoietic cells. We used a transplant model to determine whether CD34+ cells migrate from the bone marrow to Ewing's sarcoma tumors and participate in the neovascularization process that supports tumor growth. We also examined the role of VEGF165 in CD34+ cell migration. Human umbilical cord CD34+ cells were transplanted into sublethally irradiated severe combined immunodeficient mice. Seven days later, the mice were injected subcutaneously with TC71 tumor cells. Tumors were excised 2 weeks later and analyzed by immunohistochemistry. The tumor sections expressed both human VE-cadherin and mouse CD31, indicating involvement of donor-derived human cells in the tumor vessels. To determine the role of VEGF165 in the chemoattraction of CD34+ cells, we generated two VEGF165-deficient TC71 clones, a stable anti-sense VEGF165 cell line (Clone 17) and a VEGF165 siRNA-inhibited clone (TC/siVEGF(7-1)). The resulting VEGF165-deficient tumor cells had normal growth rates in vitro, but had delayed growth when implanted into mice. Immunohistochemical analysis revealed decreased infiltration of CD34+ cells into both VEGF165-deficient tumors. These data show that bone marrow stem cells contribute to the growing tumor vasculature in Ewing's sarcoma and that VEGF165 is critical for the migration of CD34+ cells from the bone marrow into the tumor.

Treatment of leukemia relapse with recombinant granulocyte-macrophage colony stimulating factor (rhGM-CSF) following unrelated umbilical cord blood transplant: Induction of graft-vs.-leukemia.

An infant with congenital leukemia in complete remission (CR1) received an unrelated donor umbilical cord blood cell transplant from a one-HLA disparate donor. The conditioning regimen consisted of thiotepa, busulfan and cyclophosphamide. GVHD prophylaxis consisted of tacrolimus and mini-methotrexate. Engraftment occurred and a bone marrow aspirate obtained on day 28 showed 100% donor cells. The post-transplant course was complicated by skin and liver GVHD, grade III, that responded to therapy with methylprednisolone, anti-thymocyte globulin and daclizumab (Zenapax), in addition to tacrolimus. A bone marrow aspirate obtained on day 187 showed relapse, with 17% blasts. The patient was then treated for 30 days with recombinant human granulocyte-macrophage colony-stimulating factor treatment (rhGM-CSF). A bone marrow aspirate obtained 17 days after the initiation of rhGM-CSF treatment showed 2% blasts. Ascites was the predominant side-effect of the rhGM-CSF treatment. The patient remains in complete remission 24 months after relapse and 30 months after transplantation. This case documents that rhGM-CSF and withdrawal of immunosuppression can induce a durable complete remission after relapse following an unrelated donor cord blood transplant.

Eradication of osteosarcoma lung metastasis using intranasal gemcitabine.

We sought to determine whether gemcitabine, a new pyrimidine antimetabolite, could inhibit the growth of human osteosarcoma cells (OS) in vitro and in vivo. Four human OS cell lines (MG-63, TE-85, SAOS-2 and SAOS-LM7) were used to assess the activity of the drug in vitro. Gemcitabine caused growth inhibition and cell death in all four cell lines as measured using the MTT and colony-forming assays (IC(50) = 6.5 nM-9 microM and 7-14 nM, respectively). Using our newly developed human SAOS-LM7 OS lung metastasis mouse model, we assessed the in vivo activity of gemcitabine given i.p. and intranasally (i.n.). Mice were treated twice weekly for 3 weeks and then once weekly for 3 weeks using either i.p. or i.n. gemcitabine starting 4 weeks after tumor cell injection. The i.p. injection, at 120 mg/kg, resulted in a decrease in lung weights and the size of the nodules. However, no significant reduction in the number of metastatic nodules was seen (control median: >200 versus gemcitabine median: 150, p = 0.084). In contrast, the number of lung metastases was significantly decreased in mice that received i.n. gemcitabine at 15 (median: 1; range: 0-115, p<0.005) and 12 mg/kg (median: 41; range: 7-163, p = 0.005) when compared with control mice (median: >200). Intranasal therapy is a non-invasive method of drug delivery and has the advantage of targeting the lung, resulting in a higher drug concentration in the tumor area. In our study, i.n. instillation of gemcitabine inhibited the growth of lung metastases at an 8- to 10-fold lower dose than that used i.p. and appeared to be more effective in eradicating OS lung nodules. Because the lung is the most common site of OS metastasis, our data suggest that i.n. gemcitabine may be a novel therapeutic approach in the treatment of OS lung metastases.