Outcomes of children, adolescents, and young adults following allogeneic stem cell transplantation for secondary acute myeloid leukemia and myelodysplastic syndromes-The MD Anderson Cancer Center experience.

We conducted a retrospective analysis of outcomes for children and young adults with sAML/sMDS who underwent HSCT at our institution. Thirty-two patients (median age 20 years) with sAML (n=24) and sMDS (n=8) received HSCT between 1990 and 2013. The median time from sAML/sMDS diagnosis to HSCT was 4.1 months (range: 1.2-27.2 months). The transplant regimens were primarily busulfan based (n=19). BM was the primary donor source (n=15). Eleven recipients were transplanted with residual disease. At a median follow-up of 62.3 months (range: 0.4-250.9 months), 14 patients had disease recurrence. Acute GVHD, grade III/IV, occurred in three patients. Causes of death were as follows: disease relapse (n=12), infection (n=2), pneumonia (n=1), pulmonary hemorrhage (n=1), acute GVHD (n=1), and graft failure (n=1). A PS of ≥90% at the time of HSCT had a significant impact on PFS (P=.02). Patients achieving pretransplant primary CR (n=8) and those with sMDS and RA (n=6) had prolonged PFS (P=.04). On multivariate analysis, shorter time to transplantation (≤6 months from diagnosis of sAML/sMDS) was associated with superior OS (P=.0018) and PFS (P=.0005).

Treatment of Severe Adult Traumatic Brain Injury Using Bone Marrow Mononuclear Cells.

Preclinical studies using bone marrow derived cells to treat traumatic brain injury have demonstrated efficacy in terms of blood-brain barrier preservation, neurogenesis, and functional outcomes. Phase 1 clinical trials using bone marrow mononuclear cells infused intravenously in children with severe traumatic brain injury demonstrated safety and potentially a central nervous system structural preservation treatment effect. This study sought to confirm the safety, logistic feasibility, and potential treatment effect size of structural preservation/inflammatory biomarker mitigation in adults to guide Phase 2 clinical trial design. Adults with severe traumatic brain injury (Glasgow Coma Scale 5-8) and without signs of irreversible brain injury were evaluated for entry into the trial. A dose escalation format was performed in 25 patients: 5 controls, followed 5 patients in each dosing cohort (6, 9, 12 ×106 cells/kg body weight), then 5 more controls. Bone marrow harvest, cell processing to isolate the mononuclear fraction, and re-infusion occurred within 48 hours after injury. Patients were monitored for harvest-related hemodynamic changes, infusional toxicity, and adverse events. Outcome measures included magnetic resonance imaging-based measurements of supratentorial and corpus callosal volumes as well as diffusion tensor imaging-based measurements of fractional anisotropy and mean diffusivity of the corpus callosum and the corticospinal tract at the level of the brainstem at 1 month and 6 months postinjury. Functional and neurocognitive outcomes were measured and correlated with imaging data. Inflammatory cytokine arrays were measured in the plasma pretreatment, posttreatment, and at 1 and 6 month follow-up. There were no serious adverse events. There was a mild pulmonary toxicity of the highest dose that was not clinically significant. Despite the treatment group having greater injury severity, there was structural preservation of critical regions of interest that correlated with functional outcomes. Key inflammatory cytokines were downregulated. Treatment of severe, adult traumatic brain injury using an intravenously delivered autologous bone marrow mononuclear cell infusion is safe and logistically feasible. There appears to be a treatment signal as evidenced by central nervous system structural preservation, consistent with previous pediatric trial data. Inflammatory biomarkers are downregulated after cell infusion. Stem Cells 2016 Video Highlight: https://youtu.be/UiCCPIe-IaQ Stem Cells 2017;35:1065-1079.

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.

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.

The development of a myeloablative, reduced-toxicity, conditioning regimen for cord blood transplantation.

Cord blood transplantation is being used with increasing frequency for patients with high-risk hematologic malignancies. Myeloablative preparative regimens provide antitumor efficacy and facilitate engraftment but are associated with higher morbidity and nonrelapse mortality rates than nonablative regimens. We evaluated 3 sequential myeloablative regimens in the cord blood transplant setting. Regimen 1 (melphalan, fludarabine, and thiotepa) produced prompt engraftment and minimal engraftment failure but was associated with a high nonrelapse mortality rate. Regimen 2 (busulfan and fludarabine) was very well tolerated but was associated with a high rate of engraftment failure and relapse. Regimen 3 (busulfan, clofarabine, fludarabine, and low-dose total body irradiation given 9 days after the chemotherapy) was associated with a low rate of engraftment failure but was logistically difficult to administer. Finally, regimen 3 that included the total body irradiation given immediately after the chemotherapy was well tolerated, with prompt engraftment and tumor control. This latter regimen appears to be effective in preliminary studies and warrants further evaluation.

Feasibility of allografting in patients with advanced acute lymphoblastic leukemia after salvage therapy with inotuzumab ozogamicin.

BACKGROUND: No highly effective salvage therapy exists for patients with relapsed acute lymphoblastic leukemia (ALL). Inotuzumab ozogamicin (IO) is a CD22 monoclonal antibody attached to calicheamycin that targets B lymphocytes in early stages of development, successfully inducing remission in patients with multiply relapsed ALL. METHODS: We describe our findings in 26 patients who received allogeneic hematopoietic stem cell transplantation (SCT) after treatment with IO between September 2010 and October 2011. RESULTS: Patients with a median age of 33 years (range, 5-70 years) received an allogeneic matched sibling donor (n = 9), matched- or 1-antigen mismatched unrelated donor (n = 16), or cord blood donor SCT (n = 1) while in complete remission (n = 23) or with active disease (n = 3). At the time of SCT, 15 patients were in complete remission without evidence of minimal residual disease (MRD) measured by multiparameter flow cytometry. Patients were heavily pretreated, including 5 patients who had received previous allogeneic SCT. Patients received a median of 3 courses of IO (range, 1-5 courses) before SCT. Seven patients are alive at a median follow-up of 13 months (range, 5-16 months), with 1-year event-free and overall survival (OS) of 22% and 20%, respectively. Patients without MRD at time of SCT had a markedly better 1-year OS of 42%. The cumulative incidence of nonrelapse mortality (NRM) at 6 months and 1 year were 40% and 60%, respectively, with 5 deaths attributed to venoocclusive disease (VOD). CONCLUSIONS: Treatment with IO allows more patients to undergo transplantation while in remission, with favorable overall survival in patients without MRD who undergo transplantation. Reduction in hepatic toxicity is needed to improve overall results.

Cord-blood engraftment with ex vivo mesenchymal-cell coculture.

BACKGROUND: Poor engraftment due to low cell doses restricts the usefulness of umbilical-cord-blood transplantation. We hypothesized that engraftment would be improved by transplanting cord blood that was expanded ex vivo with mesenchymal stromal cells. METHODS: We studied engraftment results in 31 adults with hematologic cancers who received transplants of 2 cord-blood units, 1 of which contained cord blood that was expanded ex vivo in cocultures with allogeneic mesenchymal stromal cells. The results in these patients were compared with those in 80 historical controls who received 2 units of unmanipulated cord blood. RESULTS: Coculture with mesenchymal stromal cells led to an expansion of total nucleated cells by a median factor of 12.2 and of CD34+ cells by a median factor of 30.1. With transplantation of 1 unit each of expanded and unmanipulated cord blood, patients received a median of 8.34×10(7) total nucleated cells per kilogram of body weight and 1.81×10(6) CD34+ cells per kilogram--doses higher than in our previous transplantations of 2 units of unmanipulated cord blood. In patients in whom engraftment occurred, the median time to neutrophil engraftment was 15 days in the recipients of expanded cord blood, as compared with 24 days in controls who received unmanipulated cord blood only (P<0.001); the median time to platelet engraftment was 42 days and 49 days, respectively (P=0.03). On day 26, the cumulative incidence of neutrophil engraftment was 88% with expansion versus 53% without expansion (P<0.001); on day 60, the cumulative incidence of platelet engraftment was 71% and 31%, respectively (P<0.001). CONCLUSIONS: Transplantation of cord-blood cells expanded with mesenchymal stromal cells appeared to be safe and effective. Expanded cord blood in combination with unmanipulated cord blood significantly improved engraftment, as compared with unmanipulated cord blood only. (Funded by the National Cancer Institute and others; ClinicalTrials.gov number, NCT00498316.).

High-dose infusional gemcitabine combined with busulfan and melphalan with autologous stem-cell transplantation in patients with refractory lymphoid malignancies.

We developed a new high-dose combination of infusional gemcitabine with busulfan and melphalan for lymphoid tumors. Gemcitabine dose was escalated by extending infusions at a fixed rate of 10 mg/m(2)/min in sequential cohorts, in daily, 3-dose or 2-dose schedules. Each gemcitabine dose immediately preceded busulfan (adjusted targeting area under the curve 4,000 µM/min(-1)/day × 4 days) or melphalan (60 mg/m(2)/day × 2 days). We enrolled 133 patients (80 Hodgkin lymphoma [HL], 46 non-Hodgkin lymphoma [NHL], 7 myeloma), median 3 prior regimens; primary refractory disease in 63% HL/45% NHL and positron emission tomography positive tumors at transplantation in 50% patients. Two patients died from early posttransplantation infections. The major toxicity was mucositis. The daily and 3-dose schedules caused substantial cutaneous toxicity. In contrast, the 2-dose schedule was better tolerated, which allowed us to extend the infusions from 15 to 270 minutes. Pretransplantation values of C-reactive protein, B-type natriuretic peptide, ferritin, or haptoglobin did not correlate with toxicity. Overall response and complete response rates were 87%/62% (HL), 100%/69% B large-cell lymphoma (B-LCL), 66%/66% (T-NHL), and 71%/57% (myeloma). At median follow-up of 24 months (range, 3-63 months), the event-free/overall survival rates were 54%/72% (HL), 60%/89% (B-LCL), 70%/70% (T-NHL), and 43%/43% (myeloma). In conclusion, gemcitabine/busulfan/melphalan is a feasible regimen with substantial activity against a range of lymphoid malignancies. This regimen merits further evaluation in phase II and III trials.

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.

Long-term follow-up of allogeneic hematopoietic stem cell transplantation for patients with Philadelphia chromosome-positive acute lymphoblastic leukemia: impact of tyrosine kinase inhibitors on treatment outcomes.

The introduction of tyrosine kinase inhibitors (TKI) has revolutionized therapy for patients with acute lymphoblastic leukemia (ALL) who have the Philadelphia (Ph) chromosome. A retrospective analysis was conducted on 102 adults and 11 children who received a first-matched related (n = 60), matched unrelated (n = 40), mismatched cord blood (n = 12), or haploidentical (n = 1) allogeneic hematopoietic stem cell transplantation (HSCT) for Ph-positive (Ph+) ALL in first complete remission (n = 71), second complete remission (n = 11), or with active disease (n = 31) between 1990 and 2009. Sixty-seven patients received TKI with upfront ALL therapy, and 32 patients received TKI maintenance following HSCT. With median follow-up of 5 years among survivors (range: 1.1-20.4 years), overall survival (OS) was significantly better for patients transplanted in first remission compared with HSCT in advanced disease: 43% versus 16%, P = .002. Disease stage and age at time of HSCT, the development of acute graft-versus-host disease (aGVHD), and decade of HSCT were found to significantly impact OS, progression-free survival (PFS), and nonrelapse mortality (NRM) in multivariate analyses. Allogeneic HSCT provides durable remission for patients with Ph+ ALL in first remission. Neither TKI use pre- nor post-HSCT were found to significantly impact transplant outcomes in univariate and multivariate analyses.

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.

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.

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.

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.

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.

Delayed immune reconstitution after cord blood transplantation is characterized by impaired thymopoiesis and late memory T-cell skewing.

Advances in immune assessment, including the development of T-cell receptor excision circle (TREC) assays of thymopoiesis, cytokine-flow cytometry assays of T-cell function, and higher-order phenotyping of T-cell maturation subsets have improved our understanding of T-cell homeostasis. Limited data exist using these methods to characterize immune recovery in adult cord blood (CB) transplant recipients, in whom infection is a leading cause of mortality. We now report the results of a single-center prospective study of T-cell immune recovery after cord blood transplantation (CBT) in a predominantly adult population. Our primary findings include the following: (1) Prolonged T lymphopenia and compensatory expansion of B and natural killer (NK) cells was evident; (2) CB transplant recipients had impaired functional recovery, although we did observe posttransplantation de novo T-cell responses to cytomegalovirus (CMV) in a subset of patients; (3) Thymopoietic failure characterized post-CBT immune reconstitution, in marked contrast to results in other transplant recipients; and (4) Thymopoietic failure was associated with late memory T-cell skewing. Our data suggest that efforts to improve outcomes in adult CB transplant recipients should be aimed at optimizing T-cell immune recovery. Strategies that improve the engraftment of lymphoid precursors, protect the thymus during pretransplant conditioning, and/or augment the recovery of thymopoiesis may improve outcomes after CBT.

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.

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.