Doses Of BCNU Research Articles

In Vivo HSC Gene Therapy for Hemoglobinopathies: A Proof of Concept Evaluation in Rhesus Macaques

Current gene therapy or genome editing studies for hemoglobinopathies require highly sophisticated medical facilities to perform hematopoietic stem cell collections / selections and genetic modifications. In addition, patients receive high-dose chemotherapy to facilitate engraftment of gene-modified cells. Thus, current gene therapy protocols will not be accessible to most patients suffering from hemoglobinopathies. Here we describe a highly portable and scalable approach using in vivo hematopoietic stem cell (HSC) gene therapy to potentially overcome these limitations. The central idea of our in vivo HSC gene therapy approach is to mobilize HSCs from the bone marrow, and while they circulate at high numbers in the periphery, transduce them with an intravenously injected HSC-tropic, helper-dependent adenovirus HDAd5/35++ gene transfer vector system. Transduced cells return to the bone marrow where they persist long-term. Transgene integration is either achieved by a Sleeping Beauty transposase (SB100x) in a random pattern or by homology-directed-repair into a safe genomic harbor site. Currently, an in vivo selection system (involving the mgmtP140K gene/low-dose O6BG/BCNU) is employed to achieve 80-100% marking levels in peripheral blood cells. We demonstrated safety and efficacy of our approach in mouse models for thalassemia intermedia, Sickle Cell Disease, and hemophilia A, where we achieved a phenotypic correction. We now present data in 3 rhesus macaques. We show that treatment with G-CSF/AMD3100 resulted in efficient HSC mobilization into the blood circulation and subsequent intravenous injection of the HDAd5/35++ vector system (total 1-3 x1012 vp/kg, in two doses) was well tolerated. The longest follow up thus far is 24 weeks after in vivo HSC transduction with a human-gamma-globin expressing HDAd5/35++ vector. After in vivo selection with O6BG plus low dose (10 to 20 mg/m2) of BCNU, a dose that is up to 100-fold lower than what is used for autologous transplantation protocols, gamma-globin marking in peripheral red blood cells rose to ~90% and was stable for the duration of the study (see Figure). gamma-globin levels in red blood cells were ~18% of adult alpha1-globin (by HPLC). No abnormalities in genome and transcriptome analyses of animal #1 were found at the time of scheduled necropsy. We show that a new prophylaxis regimen (dexamethasone, IL-6R, IL-1bR antagonists, saline bolus IV) was able to mitigate all side effects associated with intravenous HDAd5/35++ vector administration. Analysis of day 3 bone marrow showed 30% transduced HSCs. Vector DNA biodistribution studies demonstrated very low or absent transduction of most tissues (including testes and CNS). Analysis of bone marrow showed efficient, preferential HSC transduction and re-homing of transduced CD34+/CD90+ cells to the bone marrow. At week 4, about 5% of progenitor colony-forming cells demonstrated stable transduction with integrated vector, and this frequency increased after starting the in vivo selection. The level of human mgmtP140K mRNA expression in PBMCs also increased after in vivo selection. In summary: Using a new and optimized prophylaxis regimen intravenous delivery of HDAd5/35++ was very well tolerated without any cytokine activation. We saw efficient transduction of HSCs and efficient in vivo selection of transduced progenitors with low dose O6BG/BCNU. This is the first proof-of-concept study that in vivo HSC gene therapy could be feasible in humans without the need of high-dose chemotherapy conditioning and without the need for highly specialized medical facilities. This approach would provide a major advance for the gene therapy and genome editing field and allow the necessary portability and accessibility to reach patients in places with limited medical resources. Figure 1 Disclosures Radtke: Forty Seven INC: Consultancy. Kiem:Umoja: Membership on an entity's Board of Directors or advisory committees; Rocket Pharma: Membership on an entity's Board of Directors or advisory committees; Vor Biopharma: Membership on an entity's Board of Directors or advisory committees; Enochian: Membership on an entity's Board of Directors or advisory committees; CSL: Consultancy; Magenta Therapeutics: Consultancy; Homology Medicines: Membership on an entity's Board of Directors or advisory committees. Lieber:Ensoma, Inc: Consultancy, Research Funding.

Improved Outcomes for Relapsed/Refractory Classic Hodgkin Lymphoma Following Autologous Stem Cell Transplantation in the Era of Novel Agents

Improved Outcomes for Relapsed/Refractory Classic Hodgkin Lymphoma Following Autologous Stem Cell Transplantation in the Era of Novel Agents

Inhibitors of GLUT/SLC2A Enhance the Action of BCNU and Temozolomide against High-Grade Gliomas

Glucose transport across glioblastoma membranes plays a crucial role in maintaining the enhanced glycolysis typical of high-grade gliomas and glioblastoma. We tested the ability of two inhibitors of the glucose transporters GLUT/SLC2A superfamily, indinavir (IDV) and ritonavir (RTV), and of one inhibitor of the Na/glucose antiporter type 2 (SGLT2/SLC5A2) superfamily, phlorizin (PHZ), in decreasing glucose consumption and cell proliferation of human and murine glioblastoma cells. We found in vitro that RTV, active on at least three different GLUT/SLC2A transporters, was more effective than IDV, a specific inhibitor of GLUT4/SLC2A4, both in decreasing glucose consumption and lactate production and in inhibiting growth of U87MG and Hu197 human glioblastoma cell lines and primary cultures of human glioblastoma. PHZ was inactive on the same cells. Similar results were obtained when cells were grown in adherence or as 3D multicellular tumor spheroids. RTV treatment but not IDV treatment induced AMP-activated protein kinase (AMPKα) phosphorylation that paralleled the decrease in glycolytic activity and cell growth. IDV, but not RTV, induced an increase in GLUT1/SLC2A1 whose activity could compensate for the inhibition of GLUT4/SLC2A4 by IDV. RTV and IDV pass poorly the blood brain barrier and are unlikely to reach sufficient liquoral concentrations in vivo to inhibit glioblastoma growth as single agents. Isobologram analysis of the association of RTV or IDV and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or 4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo[4.3.0]nona-2,7,9-triene-9-carboxamide (TMZ) indicated synergy only with RTV on inhibition of glioblastoma cells. Finally, we tested in vivo the combination of RTV and BCNU on established GL261 tumors. This drug combination increased the overall survival and allowed a five-fold reduction in the dose of BCNU.

Impact of Conditioning Regimen on Outcomes for Patients with Lymphoma Undergoing High-Dose Therapy with Autologous Hematopoietic Cell Transplantation

There are limited data to guide the choice of high-dose therapy (HDT) regimen before autologous hematopoietic cell transplantation (AHCT) for patients with Hodgkin (HL) and non-Hodgkin lymphoma (NHL). We studied 4917 patients (NHL, n = 3905; HL, n = 1012) who underwent AHCT from 1995 to 2008 using the most common HDT platforms: carmustine (BCNU), etoposide, cytarabine, and melphalan (BEAM) (n = 1730); cyclophosphamide, BCNU, and etoposide (CBV) (n = 1853); busulfan and cyclophosphamide (BuCy) (n = 789); and total body irradiation (TBI)–containing treatment (n = 545). CBV was divided into CBVhigh and CBVlow based on BCNU dose. We analyzed the impact of regimen on development of idiopathic pulmonary syndrome (IPS), transplantation-related mortality (TRM), and progression-free and overall survival. The 1-year incidence of IPS was 3% to 6% and was highest in recipients of CBVhigh (hazard ratio [HR], 1.9) and TBI (HR, 2.0) compared with BEAM. One-year TRM was 4% to 8%, respectively, and was similar between regimens. Among patients with NHL, there was a significant interaction between histology, HDT regimen, and outcome. Compared with BEAM, CBVlow (HR, .63) was associated with lower mortality in follicular lymphoma (P < .001), and CBVhigh (HR, 1.44) was associated with higher mortality in diffuse large B cell lymphoma (P = .001). For patients with HL, CBVhigh (HR, 1.54), CBVlow (HR, 1.53), BuCy (HR, 1.77), and TBI (HR, 3.39) were associated with higher mortality compared with BEAM (P < .001). The impact of specific AHCT regimen on post-transplantation survival is different depending on histology; therefore, further studies are required to define the best regimen for specific diseases.

P17.59 * TRANSPLANTED MGMTP140K GENE-MODIFIED CD34+ CELLS IN PATIENTS WITH NEWLY DIAGNOSED GLIOBLASTOMA CHEMO-PROTECT BONE MARROW AND LEAD TO PROLONGED SURVIVAL

BACKGROUND: Glioblastoma is the most aggressive adult primary malignant brain tumor. It is associated with poor prognosis (median survival of 12-15 months) despite treatment. Combination chemotherapy with carmustine (BCNU) or temozolomide (TMZ) with the MGMT inhibitor O6-benzylguanine (O6BG) has been used but it has been associated with dose limiting hematopoietic toxicity. OBJECTIVE: To assess safety and efficacy of a retroviral vector encoding the O6BG-resistant MGMTP140K gene for transduction and autologous transplantation of hematopoietic stem cells (HSCs) in MGMT unmethylated, newly diagnosed GBM patients in an attempt to chemoprotect bone marrow during combination O6BG/TMZ therapy. METHODS: Seven patients have been enrolled in the first cohort of the study. Following tumor resection, patients underwent standard radiation therapy without TMZ followed by G-CSF mobilization, aphaeresis and conditioning with 600 mg/m2 BCNU prior to infusion of gene-modified cells. Post transplant, patients were treated with 28-day cycles of single dose TMZ (472mg/m2) with 48-hour intravenous O6BG (120mg/m2 bolus, followed by 30mg/m2/24h). RESULTS: The BCNU dose was nonmyeloablative. Gene marking in pre-infusion colony forming units (CFUs) was robust in most patients. Similarly, following engraftment, gene marking in white blood cells and sorted granulocytes assessed by real-time PCR was high. Patients have received up to nine cycles of O6BG/TMZ with evidence for selection of gene-modified cells and tolerable hematologic toxicity. This is significantly more cycles compared to recurrent patients receiving O6BG/TMZ in the absence of gene modified, chemoprotected cells (Quinn et al., 2005) (p < 0.05). No extra-hematopoietic toxicity has been observed thus far and all seven patients exhibited improved survival over historical controls. The longest surviving patient is 56+ months from the diagnosis without evidence of disease progression. CONCLUSIONS: These data demonstrate feasibility of achieving significant engraftment of MGMTP140K- modified cells with a well-tolerated dose of BCNU. Combination of TMZ and O6BG is well tolerated in this setting and patients with newly diagnosed GBM exhibiting unmethylated MGMT promoter achieve prolonged survivals.

Outcomes of Eam Conditioned Autologous Haematopoietic Stem Cell Transplantation for Lymphoma. A Matched Pairs Retrospective Single Centre Study Analysis.

AbstractAbstract 3088 Background:High dose chemotherapy followed by autologous stem cell rescue is superior to salvage chemotherapy alone in relapsed Hodgkin's Lymphoma (HL) and Non-Hodgkin's lymphoma (NHL). A commonly used regimen is BEAM (BCNU, Etoposide, Cytarabine and Melphalan). Unfortunately, BCNU is associated with potentially lethal pulmonary interstitial toxicity, in a dose dependent manner. Therefore, it is often omitted in conditioning regimens for patients who have pre-existing impaired lung function. We aimed to determine the outcomes of patients given this EAM (Etoposide, Cytarabine, Melphalan) schedule in our centre. Method:At our unit, from 2000–2010, 338 patients had been transplanted with BEAM conditioning and 23 patients with an EAM regimen. BEAM chemotherapy involved cumulative doses of BCNU at 300 mg/m2, etoposide at 800 mg/m2, cytarabine at 1600 mg/m2 and melphalan at 140 mg/m2. The EAM regimen was the same, except for the omission of BCNU. We wanted to compare overall survival, event free survival and transplant related mortality between the two groups of patients. A matched-pair analysis based on age and sex on a 1:1 basis was conducted. Survival rates were estimated by the Kaplan-Meier method. Differences between the survival distributions were compared with the log-rank test. Results:23 patients transplanted with an EAM regimen was matched with an equal number transplanted with a BEAM conditioning regimen. The median age was 53 in both groups. The median follow-up for patients alive was 3.2 years. Further baseline characteristics of the two groups are described in table 1. Other than a slight predominance of more advanced disease in the BEAM cohort, the two groups were evenly matched.Survival estimates were statistically significantly poorer for the EAM group compared to the BEAM group. The median overall survival for the EAM cohort was 29 months compared to 77 months (p=0.03) for the matched BEAM cohort. The median event free survival for the EAM cohort was 11 months compared to 63 months (p=0.02) for the BEAM cohort. The 100 day transplant related mortality was 8.7% for both cohorts. During the follow-up period 11 patients died of disease related causes in the EAM group compared to 6 in the BEAM group. Conclusion:Despite the prevalence of pre-existing lung disease in the EAM group of patients, the conditioning was reasonably well tolerated. Our analysis suggests that patient transplanted with an EAM regimen had an inferior survival outcome to patients treated with the standard BEAM regimen. This data provides evidence for the importance of BCNU in lymphoma control; consideration should be given to a reduction in BCNU dosage, rather than simple omission, for patients with borderline lung function results. Collaboration with other centres is warranted to determine if this experience with EAM is replicated elsewhere.Table 1:Baseline characteristics of patients analysed.BEAM (n = 23)EAM (n = 23)Age (Median, range)53, 16–7053, 16–70Sex (n)Males (14) Females (9)Males (16) Females (7)Disease and Subtype (n)HD (9) NHL (14)HD (12) NHL (11)HDNodular Sclerosing44Mixed Cellularity12Lymphocyte Predominate01Not Specified45NHLDLBCL75Follicular24Mantle Cell21T-NHL20Not specified11Stage of DiseaseI-II410III-IV1612Unknown31ChemosensitivityFirst CR10Primary Refractory34Partial response33Sensitive Relapse78Resistant Relapse34Unknown64Year of TransplantationRange2000–20102002–20102000-2005 (n)12122005-2010 (n)1111Year of diagnosis (range)1995–20091989–2010 Disclosures:No relevant conflicts of interest to declare.

Risk factors for development of pneumonitis after high-dose chemotherapy with cyclophosphamide, BCNU and etoposide followed by autologous stem cell transplant

Pneumonitis is a complication of high-dose chemotherapy and autologous stem cell transplant (HDC-ASCT) regimens containing BCNU. Our goal was to define the incidence and risk factors for pneumonitis in patients with lymphoma receiving a uniform conditioning regimen in the modern era. We studied 222 patients who received HDC-ASCT using cyclophosphamide, BCNU and VP-16 (CBV). Pneumonitis incidence was 22%, with 19% receiving systemic corticosteroid treatment and 8% requiring inpatient hospitalization for pneumonitis. Three patients died secondary to pneumonitis-related complications. The following variables were independently associated with pneumonitis: prior mediastinal radiation (odds ratio [OR] 6.5, 95% confidence interval [CI] 2.3–18.9, p = 0.0005), total BCNU dose above 1000 mg (OR 3.4, 95% CI 1.3–8.7, p = 0.012) and age less than 54 (OR 3.0, 95% CI 1.4–6.5, p = 0.0037). Increased vigilance for symptoms of pneumonitis is warranted for patients with prior mediastinal radiation and for younger patients, and dose reduction may be considered for patients who would receive greater than 1000 mg of BCNU.

BEP Versus BEAM Conditioning for Autologous Hematopoietic Cell Transplantation in Relapsed Lymphoma. A Single Center Retrospective Review of Two Contemporaneous Cohorts

Abstract 2019High-dose chemotherapy (HDC) followed by autologous hematopoietic cell transplantation (AHCT) has been shown to result in better outcomes than conventional salvage chemotherapy for treatment of relapsed Hodgkin (Lancet 2002;359:2065–71) and non – Hodgkin lymphoma patients (N Engl J Med 1995;333:1540–5).The relative efficacy of different conditioning regimens is still uncertain. Our center has had extensive experience with BEP, consisting of BCNU (600mg/m2), etoposide (2400mg/m2) and cisplatin (200mg/m2) (Lazarus HM, J Clin Oncol 1992;10:1682–9) with more than 150 patients transplanted using this conditioning regimen. We have observed it to be efficacious and associated with a low incidence of transplant-related mortality (Biol Blood Marrow Transplant 2005;11:13–22). The purpose of this analysis is to compare the outcomes of patients transplanted with BEP with a contemporaneous cohort of patients transplanted with BEAM (BCNU 300mg/m2, etoposide 800mg/m2, cytarabine 1600mg/m2, melphalan 140 mg/m2).We performed a retrospective analysis of 55 consecutive relapsed lymphoma patients who had either BEAM or BEP preparative therapy for AHCT between 2005 and 2010 at our institution. Given the potential for nephrotoxicity and ototoxicity of cisplatin, patients were selected to receive BEAM if they had previous renal dysfunction (any elevation of serum creatinine) or had previous hearing loss. All patients received corticosteroids for prophylaxis of BCNU – induced pneumonitis.The Mann – Whitney test was used for analysis of continuous variables, Fisher’s exact test for categorical data, while survival analysis was performed with the Kaplan – Meier method.Twenty-four patients received BEAM and 31 received BEP. The median age was higher in BEAM-treated patients (51 vs. 43 years, p = 0.0392). Other baseline characteristics were comparable between both cohorts: gender (male 54 vs. 58%, p = 0.791); diagnosis (NHL 75 vs. 77.4%, p = 1.000); status of disease at transplant (partial remission or worse 33.3 vs. 35.5%, p = 1.000); median number of previous therapies (2 in both groups, p = 0.51). The rate of non-renal comorbidities was higher in the BEAM cohort, but the difference was not statistically significant (45.8 vs. 32.3%, p = 0.403). The median CD34 cell dose was similar in both groups (6.252 x106 vs. 6.475 x106 CD34 cells/μL, p = 0.842).The rate of complications, including bacteremia, other infections, mucositis, diarrhea and renal dysfunction were not statistically different (Table 1). The small sample size may have prevented us from observing a statistical difference in cardiac toxicity.Table 1Complications observed after BEAM or BEP conditioning for Autologous Hematopoietic CellBEAM (%)BEP (%)Bacteremia45.835.5p = 0.580Non – bacteremic infections37.541.9p = 0.787Mucositis54.258.1p = 0.791Diarrhea79.164.5p = 0.370Increase in serum creatinine > 50%12.516.1p = 1.000Cardiac complications16.73.2p = 0.153BCNU pneumonitis4.26.4p = 1.000The median follow up time for the whole cohort was 31 months (28 vs. 34 months, p 0.267). Relapse free survival (RFS) after 36 months was 81.1% and 82.9% for BEAM and BEP, respectively (p = 0.693) (Figure 1). Overall survival at 24 months was 89.6% for BEAM and 90.8% for BEP (p = 0.371) (Figure 2). Among patients transplanted in partial response or worse, the median RFS was 57 months after BEAM and 66 months after BEP (p = 0.3173). There were no deaths in the first 100 days after transplant for both cohorts. There were no differences in the median number of days from hematopoietic cell infusion to discharge (12.5 vs. 12.0 days, p = 0.600) or achievement of ANC >500/μL (10 days for both cohorts, p = 0.415).In conclusion, BEP conditioning achieved comparable engraftment, toxicity and survival outcomes to those achieved by BEAM for treatment of relapsed lymphoma patients. BEP is therefore a valid alternative for treatment of this patient population. The BCNU dose in BEP is twice that in BEAM, but we continue to observe limited rates of BCNU – induced pneumonitis. BEP may be preferable over BEAM in patients with underlying cardiac comorbidity. Longer follow up and prospective trials will help in identifying variables that aid in the selection of patients for the most appropriate conditioning regimen. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Self-protecting core-shell magnetic nanoparticles for targeted, traceable, long half-life delivery of BCNU to gliomas

The successful delivery of anti-cancer drugs relies on the simultaneous capability to actively target a specific location, a sufficient lifetime in the active form in the circulation, and traceability and quantification of drug distribution via in vivo medical imaging. Herein, a highly magnetic nanocarrier (HMNC) composed of an Fe3O4 core and an aqueous-stable, self-doped poly[N-(1-one-butyric acid)]aniline (SPAnH) shell was chemically synthesized. This nanocarrier exhibited a high capacity for 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) drug loading. BCNU and o-(2-aminoethyl)polyethylene glycol (EPEG) were covalently immobilized on the surface of the HMNC to form a self-protecting magnetic nanomedicine (i.e., SPMNM) that could simultaneously provide low reticuloendothelial system uptake, high active-targeting, and in vivo magnetic resonance imaging (MRI) traceability. Meanwhile, the SPMNM was found to reduce the phagocytosis by macrophages and reduce the hydrolysis rate of BCNU. The high magnetization (approximately 1.2-fold higher than Resovist) of the HMNC allowed efficient magnetic targeting to the tumor. The synergetic drug delivery approach provided approximately a 3.4-fold improvement of the drug’s half-life (from 18 h to 62 h) and significantly prolonged the median survival rate in animals that received a low dose of BCNU, compared with those that received a high dose of free BCNU (63 days for those that received 4.5 mg BCNU/kg carried by the nanocarrier versus 50 days for those that received 13.5 mg of free-BCNU). This improvement could enhance the potential of magnetic targeting therapy in clinical applications of cancer treatments.

Heavy Chain Ferritin siRNA Delivered by Cationic Liposomes Increases Sensitivity of Cancer Cells to Chemotherapeutic Agents

Approximately half of all gliomas are resistant to chemotherapy, and new therapeutic strategies are urgently needed to treat this cancer. We hypothesized that disrupting iron homeostasis in glioma cells could block tumor growth, based on an acute requirement for high levels of iron to meet energy requirements associated with their rapid growth. Ferritin is best known as an intracellular iron storage protein, but it also localizes to tumor cell nuclei where it seems to protect DNA from oxidative damage and to promote transcription. In this study, we hypothesize that silencing the H-ferritin (heavy chain ferritin) gene could increase tumor sensitivity to chemotoxins. To test this hypothesis, H-ferritin siRNA was delivered to several human cancer cell lines by using cationic liposomes (C-liposome). H-ferritin siRNA decreased protein expression by 80% within 48 hours, and this decrease was associated with more than 50% decrease in the LD(50) for DNA-alkylating agent carmustine (BCNU), which is commonly used to treat glioma in clinic. In a subcutaneous mouse model of human glioma, intratumoral injections of liposomes containing H-ferritin siRNA reduced the effective dose of BCNU needed for tumor suppression by more than 50%. A plasmid supercoil relaxation assay showed that H-ferritin specifically and directly protected DNA from BCNU treatment. H-ferritin siRNA additionally seemed to increase apoptosis in glioma cells in vitro upon H-ferritin knockdown. Overall, our results illustrate how silencing H-ferritin can effectively sensitize tumors to chemotherapy and also show the ability of C-liposomes to serve as a novel in vivo delivery tool for siRNAs.

Risk Factors Associated with the Development of Pneumonitis After High-Dose Chemotherapy with Cyclophosphamide, BCNU, and Etoposide (CBV) Followed by Autologous Stem Cell Transplant

AbstractAbstract 903 Background:High-dose chemotherapy (HDC) with autologous stem cell transplant (ASCT) is a standard component of therapy for some patients with hematologic malignancies, particularly those with relapsed or refractory lymphoma. No high-dose chemotherapy regimen has been shown to be superior to another, and thus regimens are chosen based on institutional standards and toxicity profiles. Pneumonitis is a recognized complication of HDC regimens containing BCNU. There has not been a large study of uniformly-treated lymphoma patients to define the incidence and risk factors for developing pneumonitis in the modern era. Methods:We retrospectively examined the medical records of patients who were treated with HDC-ASCT at the Dana-Farber Cancer Institute and Massachusetts General Hospital Cancer Center from 2007–2009 using a regimen containing cyclophosphamide 750 mg/m2 Q12h x4d, BCNU 112.5 mg/m2 daily x4d, and VP-16 (etoposide) 200 mg/m2 Q12h x4d (CBV). Overall (OS) and progression-free survival (PFS), and the incidence of pneumonitis were determined. Univariable and multivariable analyses were performed for characteristics likely to be associated with an increased risk of pneumonitis, which was defined based on a combination of clinical, laboratory, and radiographic factors, with or without bronchoscopy. Results:222 patients were analyzed. The age range was 21–77 (median 54). 61% were male. 71% had non-Hodgkin lymphoma and 29% had Hodgkin lymphoma. The median number of prior chemotherapy regimens was 2; 65% of patients had received prior rituximab, 31% prior bleomycin, and 12% prior gemcitabine. 71% had disease involvement of the mediastinum, and 11% had received prior mediastinal radiation therapy. 43% were past or present smokers. The median follow-up among all living patients was 12 months. The total cumulative incidence of pneumonitis was 22% (49 patients), with 41 patients (19%) receiving corticosteroid treatment, and 18 patients (8%) requiring inpatient hospitalization for pneumonitis. The time range to development of pneumonitis was 26–199 days post-transplant, with a median of 50 days. There were four treatment-related deaths (1.8%): three related to pneumonitis and one related to veno-occlusive disease. On univariable analysis, age, diagnosis of Hodgkin lymphoma, prior mediastinal radiation, prior bleomycin, total BCNU dose delivered, and lack of complete remission status at the time of ASCT were associated with the development of pneumonitis. Gender, body mass index, history of smoking, mediastinal disease involvement, prior rituximab, prior gemcitabine, and pretransplant pulmonary function testing were not found to be statistically significantly different between patients with and without pneumonitis. Stepwise multivariable logistic regression analysis, excluding 31 patients without pneumonitis who had death, relapse, or censoring in the first four months post-transplant, revealed the following variables as independently associated with development of pneumonitis: prior mediastinal radiation (odds ratio 6.5, 95% CI 2.2–18.9, P=0.0005), total BCNU dose above 1000 mg (OR 3.4, 95% CI 1.3–8.7, P=0.012), and age less than 54 (OR 3.0, 95% CI 1.4–6.5, P=0.0037). One year overall survival was 92%, and progression-free survival was 71%. There were no variables, including pneumonitis, associated with PFS on multivariable analysis. Only lack of complete or partial disease response prior to ASCT was associated with inferior OS on multivariable Cox regression modeling (hazard ratio 0.2, 95% CI 0.05–0.72, P=0.01). Conclusions:Pneumonitis is relatively common after HDC-ASCT using CBV conditioning. Increased vigilance for symptoms of pneumonitis is warranted for patients with prior mediastinal radiation and for younger patients. Our data suggests an increased risk with total BCNU dose above 1000 mg, suggesting a possible threshold toxicity effect. Empiric dose reduction may be considered for patients who would receive greater than 1000 mg of BCNU, particularly if they are also younger and/or have had prior mediastinal radiation. Disclosures:No relevant conflicts of interest to declare.

Autologous Peripheral Blood Stem Cell Transplantation in Children with Refractory or Relapsed Lymphoma: Results of Children’s Oncology Group Study A5962

This prospective study was designed to determine the safety and efficacy of cyclophosphamide, BCNU, and etoposide (CBV) conditioning and autologous peripheral blood stem cell transplant (PBSCT) in children with relapsed or refractory Hodgkin and non-Hodgkin lymphoma (HL and NHL). Patients achieving complete remission (CR) or partial remission (PR) after 2 to 4 courses of reinduction underwent a granulocyte-colony stimulating factor (G-CSF) mobilized PBSC apheresis with a target collection dose of 5 × 10⁶ CD34(+)/kg. Those eligible to proceed received autologous PBSCT after CBV (7200 mg/m², 450-300 mg/m², 2400 mg/m²). Forty-three of 69 patients (30/39 HL, 13/30 NHL) achieved a CR/PR after reinduction. Thirty-eight patients (28 HL, 10 NHL) underwent PBSCT. All initial 6 patients who received BCNU at 450 mg/m² experienced grade III or IV pulmonary toxicity compared to none of the subsequent 32 receiving 300 mg/m² (P < .0001). The probability of overall survival (OS) at 3 years for all patients is 51% and for transplanted patients is 64%. The 3-year event-free survival (EFS) is 38% (45% for HL; 30% NHL). The 3-year EFS in transplanted patients is 66% (65% HL; 70% NHL). Initial duration of remission of ≥12 versus <12 months was associated with a significant increase in OS (3 years OS 70% versus 34%) (P = .003). BCNU at 300 mg/m(2) in a CBV regimen prior to PBSCT is well tolerated in relapsed or refractory pediatric lymphoma patients. A short duration (<12 months) of initial remission is associated with a poorer prognosis. Last, a high percentage of patients achieving a CR/PR after reinduction therapy can be salvaged with CBV and autologlous PBSCT.

Abstract 5529: Silencing H-ferritin by siRNA delivered by cationic liposomes increases chemotherapeutic sensitivity for treating malignant tumor

Abstract Cancer cells have a robust appetite for iron to support their rapid growth and high metabolic rates. The high levels of iron uptake require a mechanism to limit iron induced oxidative damage. This function is the responsibility of ferritin; an intracellular iron storage protein. Ferritin has been found in the nucleus of tumor cells where it appears to protect DNA from iron induced oxidative damage and promote transcription. Therefore, we hypothesized that silencing H-ferritin gene expression could increase the sensitivity of malignant tumors to radiation and chemotoxins. Potentially targets of H-ferritin disrupted iron homeostasis in tumor cells result in proving effective at negatively affecting tumor growth. To test our hypothesis, H-ferritin siRNA was delivered to U251 human astrocytoma cells, malignant peripheral nerve sheath tumor (MPNST) cells and a breast cancer cell line (MCF-7) via our cationic liposomes. Western immunoblotting analysis demonstrated that the siRNA delivered in this manner decreased H-ferritin protein expression by 70 to 80% in all three cell lines within 48 hours. The results from SRB assay displayed decrease H-ferritin was associated with a decrease in the LD50 for carmustine (BCNU) from greater than 100 µM to 40 μM in the U251 cells, 30 µM in MCF7 cells and 18 µM in MPNST cells. In addition, the results of MTS assay revealed the presence of H-ferritin siRNA was associated with a 50% increase in cell death of U251 cells at 20 Gy of radiation. The in vivo efficacy of siRNA delivered by cationic-liposomes was tested in the Athymic nude mice subcutaneous glioma tumor model. Intratumoral injections of cationic liposomes containing H-ferritin siRNA reduced the required effective dose of BCNU for tumor suppression by more than 50%. The interaction of H-ferritin with DNA was investigated in vitro by supercoil relaxation assay, which demonstrated that H-ferritin maintained DNA in a relaxed form that would promote transcription of DNA even in the presence of radiation and chemotoxins. The outcome of study for Caspase-3 activity exposed that transfection with H-ferritin siRNA was associated with activation of apoptotic caspase pathway. Overall, our data demonstrated that silencing the H-ferritin gene appears to be an effective way to sensitize tumors to radiation and chemotherapy. As importantly, we have demonstrated that siRNA can be delivered effectively by cationic liposomes in an in vivo tumor model. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5529.

Phase I/II Trial of GN-BVC, a Gemcitabine and Vinorelbine-Containing Conditioning Regimen for Autologous Hematopoietic Cell Transplantation in Recurrent and Refractory Hodgkin Lymphoma

Autologous hematopoietic cell transplantation with augmented BCNU regimens is effective treatment for recurrent or refractory Hodgkin lymphoma (HL); however, BCNU-related toxicity and disease recurrence remain challenges. We designed a conditioning regimen with gemcitabine in combination with vinorelbine in an effort to reduce the BCNU dose and toxicity without compromising efficacy. In this phase I/II dose escalation study, the gemcitabine maximum tolerated dose (MTD) was determined at 1250 mg/m 2, and a total of 92 patients were treated at this dose to establish safety and efficacy. The primary endpoint was the incidence of BCNU-related toxicity. Secondary endpoints included 2-year freedom from progression (FFP), event-free survival (EFS), and overall survival (OS). Sixty-eight patients (74%) had 1 or more previously defined adverse risk factors for transplant (stage IV at relapse, B symptoms at relapse, greater than minimal disease pretransplant). The incidence of BCNU-related toxicity was 15% (95% confidence interval, 9%-24%). Only 2% of patients had a documented reduction in diffusing capacity of 20% or greater. With a median follow-up of 29 months, the FFP at 2 years was 71% and the OS at 2 years was 83%. Two-year FFP was 96%, 72%, 67%, and 14% for patients with 0 (n = 24), 1 (n = 37), 2 (n = 23), or 3 (n = 8) risk factors, respectively. Regression analysis identified PET status pretransplant and B symptoms at relapse as significant prognostic factors for FFP. This new transplant regimen for HL resulted in decreased BCNU toxicity with encouraging FFP and OS. A prospective, risk-modeled comparison of this new combination with other conditioning regimens is warranted.

Beyond Nausea & Vomiting: Monitoring Less Common Reactions To High Dose BCNU

High dose carmustine (BCNU) is the first chemotherapy agent administered as part of the conditioning regimen BEAM +/- R for autologous blood and marrow transplantation (BMT) for lymphoma. Nausea and vomiting are common adverse drug reactions (ADR) to high dose BCNU and can be effectively controlled by administration of anti-emetics. Less common ADRs such as headache, facial flushing, oral tingling, eye problems, dizziness and allergic reaction have been reported but standard methods of prevention and/or treatment are not known.

Phase II trial of intratumoral BCNU injection and radiotherapy on untreated adult malignant glioma

DTI-015 (BCNU dissolved in ethanol) utilizes solvent facilitated perfusion (SFP) for intratumoral drug delivery. A phase II clinical trial of DTI-015 and fractionated external beam radiotherapy on newly diagnosed, malignant gliomas investigated early changes in tumour physiology and metabolism, clinical outcome and safety. Pre- and post DTI-015 injection neuro-imaging included computed tomography (CT) cerebral blood flow and volume, glucose and thallium single photon emission computed tomography (SPECT) and magnetic resonance imaging (MRI). Clinical status was determined before and after DTI-015, prior to radiotherapy and 3 monthly thereafter until progression (defined by Macdonald criteria). Primary endpoint was radiographic response. Secondary endpoints were progression free (PFS) and overall survival (OS). Twelve patients were enrolled; eight glioblastoma multiforme (GBM), four anaplastic astrocytoma (AA). Three days after DTI-015 injection, mean tumour blood flow (Paired t-test; P < 0.001) and blood volume (Paired t-test; P = 0.001) were significantly reduced. There was a significant decrease in glucose utilization (Paired t-test; P < 0.001) and thallium uptake (Paired t-test; P < 0.001) at 6 days. Tumour blood volume had a sustained reduction (Paired t-test; P = 0.001) at 26 days after DTI-015. There were two serious adverse events. Two patients with AA achieved a partial response. Median PFS was 39 weeks for AA and 27 weeks for GBM; median OS for GBM was 47 weeks and 132 weeks for AA. The imaging data forms a biological basis for understanding the effects of high dose BCNU delivered intratumorally by SFP, and suggests early effects on tumour vasculature and metabolism.

Bifunctional DNA Alkylator 1,3-Bis(2-chloroethyl)-1-nitrosourea Activates the ATR-Chk1 Pathway Independently of the Mismatch Repair Pathway

The presence of DNA damage initiates signaling through the ataxia-telangiectasia mutated kinase (ATM) and the ATM- and the Rad3-related kinase (ATR), which phosphorylate, thus activating, the checkpoint kinases (Chk) 1 and 2, which leads to cell cycle arrest. The bifunctional DNA alkylator 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) is cytotoxic primarily by inducing DNA monoadducts and ultimately, interstrand cross-links, which block DNA replication. In this study, we investigated the activation of the ATR-Chk1 pathway in response to BCNU treatment and the dependence of this response on the DNA mismatch repair (MMR) capacity. Medulloblastoma cells were exposed to low and moderate doses of BCNU, and the effects on this DNA damage signaling pathway were examined. In response to BCNU, Chk1 was found to be phosphorylated at serine 345 and exhibited increased kinase activity. Caffeine and wortmannin, which are broad-spectrum inhibitors of ATM and ATR, reduced this phosphorylation. Cell cycle analysis further revealed an accumulation of cells in the S phase in response to BCNU, an effect that was attenuated by caffeine. Small interfering RNA knockdown of ATR also reduced Chk1 phosphorylation after exposure to BCNU. However, knockdown of ATM had no effect on the observed Chk1 phosphorylation, suggesting that ATR was primarily responsible for Chk1 activation. Analysis of Chk1 activation in cells deficient in MMR proteins MutLalpha or MutSalpha indicated that the DNA damage response induced by BCNU was independent of the MMR apparatus. This MMR-independent activation seems to be the result of DNA interstrand cross-link formation.

Subcutaneous Implantation of Femur Shafts from eGFP Transgenic Mice Show Functional Hematopoietic Engraftment in MGMT Knockout Mice Selected with 1,3-Bis(2-chloroethyl)-1-Nitrosourea

There are many unanswered questions regarding the potential of stem cells and their ability to contribute to various tissues in the body. One of the difficulties in addressing issues surrounding stem cell transplantation and their contributions to various tissues is their generally poor engraftment under standard transplantation conditions. Also, transplantation via systemic delivery may result in lodging of cells in tissues where they would not normally be present, thus confusing their ability to contribute cells to those tissues. This study seeks to address the engraftment and stem cell delivery method issues by ectopically transplanting intact donor tissue as the source of stem cells and selecting for donor cells using drug resistance to encourage increased engraftment. Donor cell engraftment was followed using flow cytometry to identify eGFP+ cells in the recipient's PBL. Femur shafts from eGFP transgenic mice, which are also wildtype for the DNA repair gene O6-methylguanine-DNA methyltransferase (MGMT), were removed from donors, stripped of all muscle/fascia and other attached tissues and implanted subcutaneously into MGMT KO mice (also not expressing eGFP). Knockout of the MGMT gene in recipient mice increases the susceptibility of all tissues, especially hematopoietic stem cells to DNA damage from alkylating agents such as 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). There were 4 treatment groups, each with 2–4 immunocompetent, age and sex matched mice. Group A had femurs implanted but did not receive drug treatment, B received a 10mg/kg dose of BCNU (sub-lethal but myelosuppressive) 3 weeks post-transplant, C received a conditioning dose of BCNU 1 week prior to transplant but did not receive a post-transplant selection dose, D received both a conditioning dose and a selection dose. Two weeks post-transplant, regardless of treatment group, only rare but distinct populations of eGFP positive PBL cells (0.01–0.2%) were found. Five weeks post-transplant (2 weeks post-selection dose) there was a slight increase in eGFP positive cells 0.02–0.29% in groups A–C. Of the 3 animals in group D, 2 had a significant increase in eGFP positive PBL cells (from, <=0.2% to 1.5 and 4.8%). Two additional cohorts of animals received 4×106 whole BM cells from eGFP donor mice injected subcutaneously at the same location where the femur shafts were implanted in the other mice. One group receiving cells was treated the same as group B while the other was treated as in group D. At two weeks post-injection of cells, the animals that were not conditioned prior to receiving cells had scant eGFP+ PBL cells (0.03–0.05%). Although more distinct, the eGFP+ PBL cell populations in the animals that were conditioned prior to receiving cells were relatively small (0.08–0.16%). Five weeks after injection of the cells (2 weeks after the selection dose), mice treated as in group D demonstrated a significant population of eGFP+ PBL cells (0.4–1.8%) while the group B treated cohort had only scant eGFP+ PBL cells (0.03–0.14%). These results demonstrate that ectopically transplanted femur shafts may be used as a source of hematopoietic stem cells, that successful engraftment in immunocompetent mice requires selection for the implanted donor cells, and that donor engraftment can be increased by continued selection. We anticipate eGFP engraftment levels in the group receiving conditioning and selection will continue to increase with further selection. Long-term follow up assessments will determine if the eGFP+ donor cells from the implanted femur contribute to non-hematopoietic recipient tissues. This ectopic transplant selection model represents a novel system for interrogating the ability of stem cell source tissues to contribute to other tissues.

Metallothionein attenuates carmustine-induced oxidative stress and protects against pulmonary fibrosis in rats

The present study was carried out to evaluate the effect of exogenously administered metallothionein (MT) against carmustine (BCNU)-induced lung toxicity in rats. A total of 60 rats were randomly divided into four groups (15/group): control group in which the animals received 0.5 ml physiologic saline containing 10% ethanol (IP) weekly, MT-administered group in which rats received MT (30 micromol/kg, IP) weekly, BCNU-administered group in which rats received BCNU (5 mg/kg, IP) weekly and MT + BCNU group in which rats received weekly doses of BCNU (5 mg/kg, IP) followed 24 h later by MT (30 micromol/kg, IP). At the end of the experiment (after 6 weeks), lung histological changes, collagen staining, the activity of glutathione reductase (GR) and contents of reduced glutathione (GSH) and hydroxyproline (Hpr) in the lung as well as serum level of tumor necrosis factor-alpha (TNF-alpha) were evaluated. The obtained data revealed that BCNU induced pathological changes and markedly increased lung collagen and level of Hpr but decreased GSH content and GR activity and increased serum TNF-alpha compared to both control and MT-administered rats. Administration of MT + BCNU markedly improved histological features and decreased staining of collagen along with increased GR activity, GSH content but decreased level of Hpr in lung tissue as well as decreased serum level of TNF-alpha compared with BCNU-treated rats. Based on our results, it is possible to postulate that exogenous MT can act against BCNU-induced lung toxicity by a mechanism related, at least in part, to its ability to decrease oxidative stress and fibrosis.

Co‐expression of MGMTP140K and α‐L‐iduronidase in primary hepatocytes from mucopolysaccharidosis type I mice enables efficient selection with metabolic correction

Systemic in vivo gene therapy has resulted in widespread correction in animal models when treated at birth. However, limited improvement was observed in postnatally treated animals with mainly targeting to the liver and bone marrow. It has been shown that an O(6)-methylguanine-DNA-methyltransferase variant (MGMT(P140K)) mediated in vivo selection of transduced hematopoietic stem cells (HSC) in animals. We investigated the feasibility of MGMT(P140K)-mediated selection in primary hepatocytes from a mouse model of mucopolysaccharidosis type I (MPS I) in vitro using lentiviral vectors. We found that multiple cycles of O(6)-benzylguanine (BG)/1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) treatment at a dosage effective for ex vivo HSC selection led to a two-fold increase of MGMT-expressing primary hepatocytes under culture conditions with minimum cell expansion. This enrichment level was comparable to that obtained after selection at a hepatic maximal tolerated dose of BCNU. Similar levels of increase were observed regardless of initial transduction frequency, or the position of MGMT (upstream or downstream of internal ribosome entry site) in the vector constructs. In addition, we found that elongation factor 1alpha promoter was superior to the long-terminal repeat promoter from spleen focus-forming virus with regard to transgene expression in primary hepatocytes. Moreover, the levels of therapeutic transgene expression in transduced, enzyme-deficient hepatocytes directly correlated with the doses of BCNU, leading to metabolic correction in transduced hepatocytes and metabolic cross-correction in neighbouring non-transduced MPS I cells. These results demonstrate that MGMT(P140K) expression confers successful protection/selection in primary hepatocytes, and provide 'proof of concept' to the prospect of MGMT(P140K)-mediated co-selection for hepatocytes and HSC using BG/BCNU treatment.