Single Dose Of Cyclophosphamide Research Articles

Abstract LB-331: Effective chemoimmunotherapy with anti-TGFβ antibody and cyclophosphamide on a mouse model of breast cancer.

Abstract Elimination of CD4+FoxP3+ regulatory T cells (Tregs) has become an important strategy of cancer immunotherapy. It was reported that TGFβ is responsible for accumulation of Tregs in tumor. Thus, we treated mouse 4T1 mammary carcinoma model with 1D11 (0.1 mg/mouse), a neutralizing antibody of TGFβ(1,2,3). The treatment delayed growth of primary tumor, however, unexpectedly increased the proportion of FoxP3+ Tregs present in the intratumoral CD4 cells. In order to enhance the anti-tumor effect, 1D11 was administered with a single dose of cyclophosphamide (4 mg/mouse), a chemotherapeutic agent which was previously shown to selectively eliminate intratumoral TNFR2+ Tregs. This combination, but not the therapeutics by themselves, resulted in tumor-free long term survival of up to 80% of mice, and those tumor free mice were more resistant to re-challenge with 4T1 tumor. In order to examine the phenotype of tumor infiltrating immune cells, 4T1 tumor bearing mice were treated with 1D11 and a lower dose of cyclophosphamide (2 mg/mouse). This treatment markedly inhibited tumor growth, accompanied by a massive infiltration by IFNγ-producing T cells. Furthermore, this combination markedly decreased the number of splenic MDSCs, and increased their expression levels of MHC II and CD80. Taken together, our data show that anti-TGFβ Ab and cyclophosphamide represents a novel chemoimmunotherapeutic combination which may prove useful in the treatment of human cancer patients.(Funded by NCI Contract HHSN261200800001E) Citation Format: Xin Chen, Yuan Yang, Qiong Zhou, O.M.Zack Howard, John McPherson, Lalage Wakefield, Joost J. Oppenheim. Effective chemoimmunotherapy with anti-TGFβ antibody and cyclophosphamide on a mouse model of breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-331. doi:10.1158/1538-7445.AM2013-LB-331 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract LB-157: IL-12p70 producing dendritic cell vaccine elicits Tc1 polarized T cells and extends time to progression in metastatic melanoma.

Abstract Systemic administration of IL-12p70 has demonstrated clinical activity in cancer patients but dose-limiting toxicities have hindered its incorporation in vaccine formulations. In this proof-of-concept phase I clinical trial (NCT00683670), we report on the immunological and clinical outcomes upon vaccination of newly diagnosed stage IV melanoma patients with CD40L/IFN-γ matured IL-12p70 producing dendritic cells (mDC). HLA-A*0201+ individuals with treatment naïve metastatic melanoma were vaccinated with mDC pulsed with melanoma gp100-derived peptides, every 3 weeks by intravenous infusion for six doses, after a single dose of cyclophosphamide (300 mg/m2 iv). CT imaging was performed at baseline, week 9 and 18 for clinical assessment using RECIST. PBMC were taken weekly for immune monitoring by tetramer analysis and functional assays. Six of seven treated patients develop sustained T cell immunity to all three melanoma gp100 antigen-derived (G154, G209-2M and G280-9V) peptides, while one patient had transient immunity only to the G209-2M peptide. Three of the six immunological responders developed a radiographic response by RECIST criteria and all three individuals had a time to progression (TTP) >11.5 mo. A Cox regression analysis followed by likelihood-ratio test revealed a positive correlation (p=0.0198) between DC derived IL-12p70 and TTP. Moreover, among clinical responders, vaccine-induced gp100-specific T cells displayed a Tc1 phenotype. In contrast, a selective defect in IL-12p35 transcription was identified in clinical non-responder patient DC and gp100-specific T cells from these patients displayed the Tc2 phenotype. Incorporation of TLR3 and TLR8 agonists into the CD40L/IFN-γ maturation protocol corrected the IL-12p70 production defect in DC derived from clinical non-responder patients. These findings underscore the essential role of IL-12p70 in the development of type-1 immunity in humans with cancer and provide evidence-based rationale for incorporating IL-12p70 into the next generation of cancer vaccine formulations. Citation Format: Beatriz M. Carreno, Michelle Becker-Hapak, Alexander Huang, Megan Chan, Amer Alyasiry, Wen-Rong Lie, Rebecca L. Aft, Lynn A. Cornelius, Katherine M. Trinkaus, Gerald P. Linette. IL-12p70 producing dendritic cell vaccine elicits Tc1 polarized T cells and extends time to progression in metastatic melanoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-157. doi:10.1158/1538-7445.AM2013-LB-157

Abstract SY27-03: Biomarker-guided development of novel multi-peptide cancer vaccines - from discovery to phase lll trials.

Abstract SY27-03: Biomarker-guided development of novel multi-peptide cancer vaccines - from discovery to phase lll trials.

Antagonism of the transient receptor potential ankyrin 1 (TRPA1) attenuates hyperalgesia and urinary bladder overactivity in cyclophosphamide-induced haemorrhagic cystitis

The aim of this study was to investigate the involvement of the transient receptor potential ankyrin 1 (TRPA1) in haemorrhagic cystitis, the main side effect of cyclophosphamide-based chemotherapy. Hannover female rats received intraperitoneal (i.p.) injection of cyclophosphamide (three doses of 100mg/kg, every other day, in a total of five days). This treatment was followed by the treatment with TRPA1 antagonist HC 030031 (50mg/kg, p.o.). The threshold for hindpaw withdrawal or abdominal retraction to von Frey Hair and the locomotor activity were measured. The treatment with the TRPA1 antagonist HC 030031 significantly decreased mechanical hyperalgesia induced by cyclophosphamide without interfere with locomotor activity. Urodynamic parameters were performed by cystometry 24h after a single treatment with cyclophosphamide (200mg/kg, i.p.) in control and HC 030031 treated rats. Analyses of the urodynamic parameters showed that a single dose of cyclophosphamide was enough to significantly increase the number and amplitude of non-voiding contractions and to decrease the voided volume and voiding efficiency, without significantly altering basal, threshold or maximum pressure. The treatment with HC 030031 either before (100mg/kg, p.o.) or after (30mg/kg, i.v.) cyclophosphamide inhibited the non-voiding contractions but failed to counteract the loss in voiding efficiency. Our data demonstrates that nociceptive symptoms and urinary bladder overactivity caused by cyclophosphamide, in part, are dependent upon the activation of TRPA1. In this context, the antagonism of the receptor may be an alternative to minimise the urotoxic symptoms caused by this chemotherapeutic agent.

Haploidentical bone marrow transplants for haematological malignancies using non‐myeloablative conditioning therapy and post‐transplant immunosuppression with cyclophosphamide: results from a single Australian centre

To demonstrate safety and efficacy of haploidentical bone marrow transplantation with non-myeloablative conditioning and high-dose post-transplant cyclophosphamide in adult patients with leukaemia or lymphoma. Human leukocyte antigen haploidentical bone marrow transplantation is a treatment option in patients with haematological malignancies who have no available human leukocyte antigen-matched donor but is limited by conditioning regimen toxicity, graft failure, relapse and graft-versus-host disease (GvHD). Twelve patients, median age of 51 years, underwent transplantation with T cell replete bone marrow from a haplotype-matched relative. The conditioning regimen consisted of cyclophosphamide, fludarabine and low-dose total body irradiation. Post-transplant immunosuppression consisted of a single dose of cyclophosphamide 50 mg/kg on day 3, followed by oral tacrolimus and mycophenolate mofetil. Outcomes reported are overall survival, engraftment and chimerism, toxicity, and clinical outcome. All patients had neutrophil recovery (median 14.5 days), and 11 of 12 had platelet engraftment (median 17 days). Two patients had autologous reconstitution. Seven of nine assessable patients had complete donor chimerism. Four patients had grades II-III GvHD, and none had grade IV GvHD. Four patients developed limited stage chronic GvHD. Five patients with acute myeloid leukaemia relapsed. Two patients died of nonrelapse causes, both from other malignancies, and five patients remain alive and relapse free. Median overall survival was 324 days (range 88-1163). This regimen is feasible and well tolerated in older patients with high-risk leukaemia or lymphoma, with minimal short-term toxicity and low rates of GvHD. The proportion of disease-free survivors indicates a graft-versus-malignancy effect is present in survivors.

Ranolazine-Induced Severe Bladder Hypotonia; Fatal Varicella-Zoster Vasculopathy Associated with Adalimumab Therapy; SIADH Induced by a Single Dose of Cyclophosphamide; Tetany, Hypomagnesemia, and Proton Pump Inhibitors; Unnecessary Surgery for Acute Abdomen Caused by ACE Inhibitor Use; Atazanavir-Associated Renal Stones and Biliary Stones; Drug-Induced Hallucinations Associated with Amantadine and Tizanidine

The purpose of this feature is to heighten awareness of specific adverse drug reactions (ADRs), discuss methods of prevention, and promote reporting of ADRs to the US Food and Drug Administration's (FDA's) MEDWATCH program (800-FDA-1088).

Anti-CD45 Radioimmunotherapy Facilitates Donor Engraftment and Prolongs Survival in the Absence of TBI Prior to Haploidentical Bone Marrow Transplantation in a Disseminated Murine Leukemia Model

AbstractAbstract 4101 Background:Despite the curative promise of hematopoietic cell transplantation (HCT), many patients with hematologic malignancies relapse and others may not proceed to HCT due to the unavailability of a matched donor. Toxicities remain high with HCT, due in part to the administration of non-specific therapies such as total body irradiation (TBI) as part of preparative regimens. We aim to overcome these limitations by replacing TBI with anti-CD45 radioimmunotherapy (RIT) for haploidentical HCT to deliver radiation directly to leukemic cells while sparing normal organs and minimizing non-specific toxicities. Methods:We established an initial TBI HCT regimen in B6SJLF1/J mice (H-2Db haplotype) conditioned with fludarabine (FLU, days -6 to -2), followed by TBI (250, 500, 750 cGy; day -1). The mice then received 15 million donor (CB6F1/J, H-2Dd) BM cells (day 0), followed by cyclophosphamide (CY) for graft-versus-host disease (GvHD) prophylaxis (day +2). Subsequent RIT HCT studies involved B6SJLF1/J mice conditioned with and without fludarabine (FLU) and escalating doses (200–400 μCi) of 90Y-anti-CD45 Ab (30F11) RIT without TBI, followed by infusion of haploidentical BM cells from CB6F1/J mice and a single dose of cyclophosphamide (CY) 2 days after HCT. Chimerism studies were performed using flow cytometric analysis to assay for engraftment of donor CD8+ cells. Therapeutic studies were performed in B6SJLF1/J mice given 105 syngeneic leukemia cells via tail vein (day -5), followed by 200 or 400 μCi 90Y-30F11 (day -3), and 1.5 × 107 BM donor cells (day 0) and two doses of CY (days -2 and +2) without FLU. Results:Using this model we have demonstrated that mixed chimerism was established in mice transplanted with TBI or escalating doses (200–400 μCi) of 90Y-30F11 RIT followed by injection of haploidentical BM donor rescue cells. TBI-based HCT showed that chimerism as determined by flow cytometric analysis for donor CD8+ cells was TBI dose-dependent; mice receiving ≥500 cGy were fully chimeric 4 weeks post-HCT, and persisted ≥12 months. RIT-based HCT also revealed mice with mixed chimerism, with up to 89% of donor CD8+ cells 1 month after HCT. Elimination of FLU from the conditioning regimen did not significantly decrease chimerism, as mice transplanted without FLU showed up to 70% donor CD8+ cells 1 month after HCT. Subsequent RIT experiments in B6SJLF1/J mice harboring AML were treated with escalating doses of 90Y-30F11 prior to HCT without FLU. Mice treated with anti-CD45 RIT using 200 μCi and 400 μCi of 90Y-30F11 had a median overall survival (OS) of 73 (p<0.001) and 107.5 (p=0.0015) days, respectively, compared to untreated leukemic control mice which had a median OS of 34 days after HCT (p values by Log rank (Mantel-Cox) testing)(Figure). Two mice in the 400 μCi-90Y-30F11 group were euthanized on day 3 for excessive weight loss, without gross histology abnormality in kidneys or liver. Conclusion:These studies suggest that anti-CD45 RIT in the absence of TBI and FLU prior to haploidentical HCT can lead to establishment of mixed chimerism. Moreover, this anti-CD45 RIT in combination with haploidentical HCT can lead to improvement in survival for mice with AML. These results suggest that clinical studies with anti-CD45 RIT in lieu of TBI and FLU in a haploidentical HCT regimen should be considered for further investigation. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Synergestic effect of lycopene and Melatonin against the genesis of oxidative stress induced by cyclophosphamide in rats

Cyclophosphamide (CP) is an alkylating agent that has been considered effective for cancer treatment. Lycopene, the pigment in tomato fruits, has beneficial effect in the treatment of some diseases. The goal of this study is to evaluate the protective effect of lycopene alone or combined with melatonin (Mel) in inhibiting the oxidative stress and toxic effect of CP in rats. Five groups of rats were included in this study; Group I served as the control. Rats in groups II-V were administrated with single dose of CP (150 mg/kg B.W) interperitoneally for 3 days. On the same day of CP administration, the rats in group III were fed a diet supplemented with lycopene (50 mg/kg of diet), rats in group IV were administered with a dose of 2.5 mg Mel/kg body weight (bw) injected subcutaneously and rats in group V were supplemented with lycopene and a dose of 2.5 mg Mel/kg bw injected subcutaneously. After 15 days the blood samples were collected. Results obtained showed that CP exerted its toxic effect by increasing the free radicals and reactive oxygen species that causes lipid peroxidation and cell damage, and this in turn is detected by elevation in nitric oxide (NO) and malondialdehyde (MDA), while the activities of antioxidants enzymes including (superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx)) were significantly decreased as compared with the control rats. The combined treatment (Lyco + Mel) group showed potential reduction in these parameters more than those treated with lyco alone. The activities of SOD, CAT and GPx were found significantly high than lyco alone treated rats. A positive significant correlation between NO and MDA (r = .81). In conclusion, these results suggested that supplementation of diet with lycopene and Mel provided antioxidant defense with strong chemopreventive activity against Cp-induced cytotoxicity.

The effects of bestatin on humoral response to sheep erythrocytes in non-treated and cyclophosphamide-immunocompromised mice

The effects of bestatin on humoral immune response to sheep erythrocytes (SRBC) and restoration of the response impaired by a single cyclophosphamide dose (350 mg/kg) were tested on mice. Bestatin (at doses of 10, 1, and 0.1 mg/kg) was administered intraperitoneally (i.p.) 5 or 10 times. The pharmacological immunosuppression was induced by a single i.p. injection of cyclophosphamide (350 mg/kg) administered 24 h before the first bestatin dose. The mice were immunized i.p. with SRBC 24 h after the last dose of bestatin. It was found that multiple administration of bestatin at all three doses potentiated the humoral response to SRBC in non-treated mice, resulting in an increased number of plaque-forming cells (PFC) and 2-mercaptoethanol (2-ME)–resistant anti-SRBC antibodies. However, five times administration of bestatin at the doses under investigation caused further decreases in total anti-SRBC hemagglutinins. A single injection of cyclophosphamide (350 mg/kg) suppressed humoral response of mice to the antigen. Administration of bestatin after pharmacological immunosuppression partially prevented the suppressive action of cyclophosphamide in the in vivo model of the humoral immune response to SRBC. The protective action of bestatin was both dose- and schedule-dependent. Ten times’ exposure to a bestatin dose of 0.1 mg/kg after a high cyclophosphamide dose partially reduced the suppressive effect of this drug on humoral response of SRBC-immunized mice, increasing PFC on days 4 and 7 after immunization, which coincided with restored ability of the lymphocytes to produce the 2-ME–resistant hemagglutinins on day 7 and the total anti-SRBC hemagglutinins on day 14 after priming.

The Protective Effects of Spirulina in Cyclophosphamide Induced Nephrotoxicity and Urotoxicity in Rats

To evaluate the role of Spirulina, a blue-green algae with antioxidant properties in the protection of cyclophosphamide-induced nephrotoxicity and hemorrhagic cystitis in rats. The control group (C) was sacrificed 24 hours after being given a single dose of saline intraperitoneally (150 mg/kg) on the seventh day of the experiment. The rats in the second group (CP) were sacrificed 24 hours after being given a single dose of cyclophosphamide, intraperitoneally (150 mg/kg) on the seventh day of the experiment. Spirulina was administered to the third group (SP+CP) orally (1000 mg/kg bw/day) for 7 days and a single dose of cyclophosphamide was injected intraperitoneally (150 mg/kg) on the seventh day of the experiment. At the eighth day of the experiment, malondialdehyde, superoxide dismutase, and catalase levels in renal and urinary bladder tissues were measured. Histomorphology in urinary bladder, apoptosis by caspase 3 immunostaining, and TUNEL assay in kidney were also evaluated. Tissue levels of malondialdehyde in the SP+CP group were significantly lower versus CP group (P < .05). Tissue levels of superoxide dismutase and catalase in the SP+CP group were significantly higher vs the CP group (P < .05). The histomorphologic alteration in urinary bladder in the SP+CP group was significantly lower vs that in the CP group. In the kidney, apoptosis in the SP+CP group as shown with TUNEL assay and immunohistochemistry was significantly lower vs that in the CP group (P < .05). Pretreatment with Spirulina protects the rats from cyclophosphamide-induced nephro-urotoxicity via its antioxidant and anti-apoptotic properties.

Multipeptide immune response to cancer vaccine IMA901 after single-dose cyclophosphamide associates with longer patient survival

IMA901 is the first therapeutic vaccine for renal cell cancer (RCC) consisting of multiple tumor-associated peptides (TUMAPs) confirmed to be naturally presented in human cancer tissue. We treated a total of 96 human leukocyte antigen A (HLA-A)*02(+) subjects with advanced RCC with IMA901 in two consecutive studies. In the phase 1 study, the T cell responses of the patients to multiple TUMAPs were associated with better disease control and lower numbers of prevaccine forkhead box P3 (FOXP3)(+) regulatory T (T(reg)) cells. The randomized phase 2 trial showed that a single dose of cyclophosphamide reduced the number of T(reg) cells and confirmed that immune responses to multiple TUMAPs were associated with longer overall survival. Furthermore, among six predefined populations of myeloid-derived suppressor cells, two were prognostic for overall survival, and among over 300 serum biomarkers, we identified apolipoprotein A-I (APOA1) and chemokine (C-C motif) ligand 17 (CCL17) as being predictive for both immune response to IMA901 and overall survival. A randomized phase 3 study to determine the clinical benefit of treatment with IMA901 is ongoing.

CD40 ligand/interferon-γ matured DC immunization with gp100 antigen HLA class I A *0201 restricted peptides in patients with newly diagnosed metastatic melanoma.

2525 Background: CD40L/IFN-γ matured Dendritic Cells (DCs) produce IL-12 and are potent antigen-presenting cells for naïve resting T cells. We sought to determine the magnitude and kinetics of CD8+ T cell growth in patients receiving autologous CD40L/IFN-γ matured DC and identify biomarkers associated with clinical outcome. Methods: A phase I clinical trial (NCT00683670) incorporating CD40L/IFN-γ for the ex vivo maturation of autologous DCs pulsed with three well characterized gp100 melanoma antigen derived peptides (G154, G209-2M, G280-9V) was initiated with enrollment from 2008-11 at a single center. HLA-A*0201+ individuals with treatment naïve metastatic melanoma were immunized every 3 weeks by intravenous infusion for six doses after a single dose of cyclophosphamide (300 mg/m2 iv). CT imaging was performed at baseline, week 9 and 18 for clinical assessment using RECIST. Responding patients were eligible for maintenance doses every 2-4 months. PBMC were taken weekly for immune monitoring by tetramer analysis and functional assays. DC preparations were characterized to assess for biomarkers of response. Results: 10 patients were screened. Among the 7 treated patients, there were 3 confirmed responses (independently verified), including one durable CR &gt;3 years and 2 PR. Three patients had rapid disease progression and received only 3 doses. Four patients (1 CR, 2 PR, 1 PD) received 6 or more vaccine doses. No SAEs were noted. There was no correlation between tumor volume and response. Using pre-specified immune response criteria, 6 (86%) treated patients developed CD8+ T cell immunity to all three peptides as assessed by tetramer analysis. The vaccine-induced T cells from all 6 individuals were polyfunctional and killed gp100+, HLA-A2+ human melanoma targets in a standard 51Cr release assay. IL-12 production by DCs correlated with TTP (p=0.0198, likelihood ratio test) but not OS (p=0.08). Conclusions: Weekly immune monitoring reveals the rapid onset of CD8+ T cell immunity against gp100 among the responder patients. This is the first DC vaccine clinical trial in melanoma to demonstrate a correlation of IL-12 production and TTP.

Abstract 5365: Prolonged survival of patients with advanced renal cancer responding to multi-peptide vaccine IMA901 after single-dose cyclophosphamide

Abstract Translational research and the clinical development of therapeutic cancer vaccines requires stronger scientific rationalization. Here we demonstrate how immune response markers as well as biomarkers defining the immune regulatory environment were utilized as guiding tools from discovery to advanced clinical trials of IMA901, a novel therapeutic vaccine for the treatment of renal cell carcinoma (RCC). IMA901 consists of multiple tumor-associated peptides (TUMAPs) confirmed to be naturally presented in human RCC tissue by mass spectrometry, selected using differential transcriptomics and preclinically validated by systematic analysis of immunogenicity with artificial antigen-presenting cells. Two consecutive independent clinical studies in a total of 96 HLA-A*02+ advanced/metastatic RCC patients were conducted. The phase I study revealed that T-cell responses to multiple IMA901 antigens were significantly associated with disease control and negatively associated with the presence of FoxP3+ regulatory T cells (Tregs). The subsequent randomized phase II study demonstrated that pre-treatment with a single low dose of cyclophosphamide (Cy) reduced Treg frequencies and prolonged overall survival (OS) in patients who mounted an immune response to the IMA901 vaccine. Additionally, T-cell responses to multiple IMA901 antigens were again associated with clinical benefit. Furthermore, a comprehensive prognostic and predictive biomarker program was conducted. Among cellular biomarkers, highly significantly elevated levels of myeloid-derived suppressor cells (MDSC), IL-17-/IL-10-secreting T cells and dysfunctional T cells in RCC patients vs. healthy individuals were found. Two MDSC populations (CD14+ HLA-DR- and CD14- CD11b+ CD15+) were significantly negatively associated with survival in vaccinated RCC patients. Interestingly, both MDSC populations were also found to be negatively associated with OS in an independent trial in colorectal cancer patients (N=79) implying a broader role for these MDSC species. Additionally, among over 300 serum biomarkers tested, apolipoprotein A-I (ApoA1) and the chemokine CCL17 were found to be predictive for both immune responses to IMA901 and survival of the RCC patients. The knowledge acquired in these trials was used to design a randomized phase III study. In this ongoing study, IMA901 is combined with the tyrosine kinase inhibitor sunitinib based on the findings that sunitinib downmodulates the two MDSC populations described above. Furthermore, in this phase III study, the relevance of ApoA1/CCL17 will be explored by prospectively defined subgroup analyses. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5365. doi:1538-7445.AM2012-5365

Using clinically approved cyclophosphamide regimens to control the humoral immune response to oncolytic viruses

Oncolytic viruses can be neutralized in the bloodstream by antiviral antibodies whose titers increase progressively with each exposure, resulting in faster virus inactivation and further reductions in efficacy with each successive dose. A single dose of cyclophosphamide (CPA) at 370 mg m(-2) was not sufficient to control the primary antiviral immune responses in mice, squirrel monkeys and humans. We therefore tested clinically approved multidose CPA regimens, which are known to kill proliferating lymphocytes, to determine if more intensive CPA therapy can more effectively suppress antiviral antibody responses during virotherapy. In virus-susceptible mice, primary antibody responses to intravenously (i.v.) administered oncolytic measles virus (MV) or vesicular stomatitis virus (VSV) were partially or completely suppressed, respectively, by oral (1 mg × 8 days) or systemic (3 mg × 4 days) CPA regimens initiated 1 day before virus. When MV- or VSV-immune mice were re-challenged with the respective viruses and concurrently treated with four daily systemic doses of CPA, their anamnestic antibody responses were completely suppressed and antiviral antibody titers fell significantly below pre-booster levels. We conclude that the CPA regimen of four daily doses at 370 mg m(-2) should be evaluated clinically with i.v. virotherapy to control the antiviral antibody response and facilitate effective repeat dosing.

Effects of cyclophosphamide myelosuppression in adult dogs with latent canine herpesvirus-1 infection

Latent canine herpesvirus-1 (CHV-1) infection is common in domestic dogs, but triggers for viral reactivation and recrudescent CHV-1 disease are poorly understood. Cyclophosphamide is a potent immunosuppressive and myelosuppressive agent used for the therapy of a variety of neoplastic and immune-mediated canine disorders. Cyclophosphamide (200mg/m2) was administered to mature dogs latently infected with CHV-1 to determine its potential to induce recurrent CHV-1 disease and viral shedding. Non-infected dogs and dogs recovered from experimental primary ocular CHV-1 infection with experimentally confirmed latent CHV-1 infection were divided into groups and administered cyclophosphamide or placebo. Dogs were monitored for myelosuppression and viral reactivation for 28days using clinical and virological outcome measures. Clinical ophthalmic and in vivo ocular confocal microscopic examinations were performed at intervals. Samples were collected for CHV-1 polymerase chain reaction (PCR), CHV-1 virus neutralizing (VN) antibody, and hemogram assays. Myelosuppression (i.e., decreased total leukocyte, segmented neutrophil, and erythrocyte counts) was detected on study day 7 in dogs administered cyclophosphamide, but not dogs administered placebo. There were no abnormalities suggestive of recurrent CHV-1 ocular disease during clinical ophthalmic or in vivo confocal microscopic examination in any dogs during the study. Ocular CHV-1 shedding was not detected by PCR and CHV-1 VN titers remained stable in all dogs. Following study conclusion, the presence of reactivatable latency was reconfirmed in the infected dogs by administering systemic prednisolone. Myelosuppression elicited by a single dose of cyclophosphamide does not result in detectable recurrent ocular CHV-1 infection in adult dogs with experimentally induced latent CHV-1 infection.

Effect of cyclophosphamide exposure on the migration of primordial germ cells in rat fetuses

Effect of a single dose of cyclophosphamide on migration of the primordial germ cells (PGC), when they are about to reach gonadal ridge was investigated histochemically by staining for alkaline phosphatase. This may throw some light on the fate of gonadal ridge when exposed to the drug itself or its breakdown products such as acrolein, which is present as an environmental pollutant. Twelve pregnant Charles foster rats were divided in to control and treatment groups and kept in separate cages. In the experimental group, Cyclophosphamide 20 mg/kg/body weight was injected intraperitoneally on day 12 of gestation. Transverse sections of fetuses collected on day 16 of gestation were stained for alkaline phosphatase activity. Outcome of the study was analysed by scanning the photomicrographs and represented by photomicrographs. An unique finding in experimental group in the gonadal ridge consisted of homogeneously distributed pale staining cells. The gonadal ridge-mesonephros junction showed a single big cluster of the PGC. Under higher magnification, the PGC could be identified by oval or circular shape with well-defined cell membranes and very distinct dark brown staining. There were no signs of degeneration or disintegration of these cells. Cyclophosphamide exposure led to failure of PGC to spread inwards from the gonadal ridge-mesonephros junction giving rise to a situation so far not reported in literature. The presented phenomenon will result in improper development of the gonads leading to infertility in an affected individual in future generation (Fig. 4, Ref. 18).

Randomized Phase II Study of BLP25 Liposome Vaccine (L-BLP25) in Patients with Multiple Myeloma

Abstract 2927The standard of care for patients (pts) with asymptomatic multiple myeloma (MM) stage I/II or a stable response after chemotherapy is “watchful waiting”, with conventional agents failing to demonstrate benefits as early or maintenance therapy. We report data from a randomized, open-label, phase II study of BLP25 liposome vaccine (L-BLP25, Stimuvax®), a therapeutic cancer vaccine (TCV) targeting the mucin 1 (MUC1) antigen, in pts with previously untreated, slowly progressive, asymptomatic MM I/II or MM II/III in stable response/plateau phase following anti-tumor therapy.Pts were randomized to L-BLP25 and either a single (Group [Gp] A) or multiple (Gp B) low dose(s) of cyclophosphamide (CP). In both Gps, L-BLP25 (930 μg) was administered weekly for 8 wk, followed by maintenance vaccination every 6 wk until disease progression requiring anti-tumor therapy. A single dose of CP (300 mg/m2, maximum total dose 600 mg) was given 3 days before the first L-BLP25 dose and, in Gp B, also prior to the vaccination at wk 5 and each maintenance phase vaccination.The primary objective was to investigate the immune response to L-BLP25. A specific induced immune response to MUC1 was defined as ≥2-fold increase over baseline and no peptide control in an IFN- γ ELISpot, lymphoproliferation or intracellular IFN- γ FACS assay following short-term in vitro stimulation of PBMCs with MUC1-derived peptides, on ≥2 assessments. Secondary objectives included safety/tolerability, quality of the immune response, linkage of immune response to HLA restriction, clinical effects, time to progression and time to anti-tumor therapy. Median data are presented.Thirty-four pts (age: 64 years; 15 male) were randomized and received treatment (Safety Analysis [SA] set; A/B = 17/17) of which 32 (17/15) pts met the pre-specified criteria for immunologic analysis (Immunological Diagnostic Analysis [IDA] set). The duration of MM (SA set) was 34 vs 37 months (Gp A vs B) at study entry. The proportion of pts with untreated stage I/II vs previously treated stage II/III MM was 29 vs 71% (Gp A) and 47 vs 53% (Gp B). Of the previously treated pts, 83% (Gp A) and 100% (Gp B) had received high-dose chemotherapy with autologous stem cell transplantation.In this analysis, all pts had reached ≥50 wk or had discontinued study treatment. Treatment duration was 54 vs 87 wk (Gp A vs B), with 15 vs 21 L-BLP25 vaccinations and 1 vs 11 CP infusions (SA set). Cumulative CP dose (first 50 wk) was 297 vs 1769 mg/m2 (Gp A vs B), corresponding to a relative dose intensity of 100 and 97%.As in previous studies, spontaneously induced specific MUC1 immune responses were seen frequently pre-vaccination (baseline Gp A/B = 59/47%). Specific induction/augmentation of the MUC1 response was seen in 53% of pts following L-BLP25 treatment with no difference between Gps A and B. Rates of induction/augmentation following vaccination were similar for pts with vs without a spontaneous (pre-vaccination) immune response. First immune responses occurred early (≤9 wk) in the course of treatment. Assessments of cytokines will be presented.Objective clinical responses (Bladé criteria) were not seen, and were not anticipated given the aim of TCVs is to stabilize disease rather than induce a response according to established chemotherapy criteria. In a preliminary analysis, reduction in the slope of on-study M-protein concentration over time compared with pre-study data was seen in 13 of 30 pts (10/13 previously untreated and 3/17 previously treated; updated analysis will be presented). There was no association between presence of a treatment-induced MUC1 response and on-study changes in M-protein (area under the curve at wk 26 [AUC26]; IDA set). However, on-study M-protein AUC26 was significantly lower in pts without vs with a spontaneous pre-vaccination MUC1 response (pooled data for both groups: AUC26 = -5.2 [n=13] vs 16.2 [n=16], p=0.015). However, these data should be interpreted with caution given the small number of pts.Treatment was generally well tolerated. One possibly treatment-related fatal event of encephalitis occurred in Gp B.In summary, L-BLP25 led to MUC1-specific immune responses in a large proportion of MM pts and was associated with emerging clinical effects on M-protein concentration over time. Our data suggest that pts with previously untreated, early-stage disease may be the most likely to benefit from L-BLP25 vaccination. Further investigation of L-BLP25 in MM is warranted. Disclosures:Off Label Use: The abstract reports the results of a Phase II investigative study of L-BLP25 in multiple myeloma. L-BLP25 is a therapeutic cancer vaccine targeting mucin 1, which is widely expressed on common cancers. Österborg:Merck GmbH: Research Funding. Forssmann:Merck Serono S.A., 9 Chemin des Mines, CH-1202 Geneva, Switzerland: Employment. Senger:Merck KGaA, Darmstadt, Germany: Employment. Schröder:Merck KGaA, Darmstadt, Germany: Employment. Mellstedt:Merck-Serono: Honoraria, Research Funding.

LICC: L-BLP25 in patients with colorectal carcinoma after curative resection of hepatic metastases—A randomized, double-blind, placebo-controlled, multicenter, multinational, phase II trial.

TPS166 Background: Distant metastases limited to the liver occur in 15-20% of all patients initially diagnosed with colorectal cancer. Complete surgical resection represents the only curative option. The 5-year survival after R0-resection of liver metastases ranges between 28 and 39%. Unfortunately, recurrence rates peak up to 70% in long-term follow-up. Adjuvant chemotherapeutic protocols did not reveal significant improvement of the situation. The primary objective of the LICC trial is to determine whether the therapeutic cancer vaccine L-BLP25 extends recurrence-free survival (RFS) time over placebo in colorectal cancer patients after resection of hepatic metastases. L-BLP25 targets the MUC1 glycoprotein, which is highly expressed in hepatic metastases of colorectal cancer. In a phase IIB trial, L-BLP25 showed acceptable tolerability and a trend toward longer survival in patients (pts) with stage IIIB locoregional NSCLC. Methods: This is a planned multinational, phase II, multicenter, randomized, double-blind trial. Planned sample size is 159 pts from 20 centers in 3 countries; accrual is planned to start in Q3 2011. Pts with colorectal adenocarcinoma after complete resection of the primary tumor and having experienced a curative-intent resection of all hepatic metastases (R0/R1, metachronous/synchronous, <6 weeks) are eligible. Pts will be randomized 2:1 to receive either L-BLP25 or placebo. Those in the L-BLP25 arm receive a single dose of cyclophosphamide (CP) (300 mg/m2) 3 days before first L-BLP25 dose, then primary treatment with sc L-BLP25 930 μg once weekly for 8 weeks, followed by sc L-BLP25 930 μg maintenance doses at 6-week (years 1&2) and 12-week (year 3) intervals until recurrence. In the control arm, CP is replaced by NaCl solution and L-BLP25 by placebo. Stratified: R0 vs. R1. Primary endpoint: RFS time between treatment groups. Secondary endpoints: overall survival (OS) time, safety, tolerability, RFS/OS in MUC-1 positive cancers. Exploratory immune response analyses are planned. Evaluation of primary endpoint is planned for Q3 2016.

Ultrastructural changes in the rat kidney after single dose of cyclophosphamide—Possible roles for peroxisome proliferation and lysosomal dysfunction in cyclophosphamide-induced renal damage

Electron microscopy was used to examine changes in the subcellular organelles of the rat kidney at different time intervals after a single exposure to cyclophosphamide (CP). The morphological changes were studied at different time points (6 hrs, 16 hrs and 24 hrs) after a single-dose administration of CP. Six rats were killed at each time intervals after the administration of CP. Saline-treated rats served as controls. CP administration resulted in alterations in various subcellular organelles including peroxisomes, lysosomes, mitochondria, and the endoplasmic reticulum (ER) of the renal tubular epithelium as well as damage to the glomerulus. The basement membrane of the glomerulus was thickened. Many podocytes were destroyed. The nucleoplasm of the endothelial cell showed fewer granularities. The tubules were distorted and the brush border was destroyed. Two striking features in the renal tubular cells are increase in number and size of the peroxisomes (peroxisome proliferation) and decrease in the number of lysosomes. The mitochondria were elongated and the number was increased in the tubules of CP-treated rats. The ER was dilated. Cell necrosis was also seen. This study is an evidence of changes in morphology of rat kidney after induction of renal damage by a single dose of CP. Since transmission electron microscopy is the highest magnification tool at present, it can be useful in estimating the degree of injury and outcome of alternative treatment strategies in the management of CP-induced renal damage after establishing a scoring system.

Analysis of micronuclei, histopathological changes and cell proliferation in nasal epithelium cells of rats after exposure to formaldehyde by inhalation

The frequencies of micronuclei (MN), histopathological changes and cell proliferation were determined in the nasal epithelium of male Fischer-344 rats after exposure to formaldehyde (FA) by whole-body inhalation for four weeks (6 h/day, 5 days/week). Groups of 12 rats each were exposed to the target concentrations of 0, 0.5, 1, 2, 6, 10 and 15 ppm. The micronucleus test (MNT) was performed with nasal epithelial cells prepared from six animals per group. Two thousand cells per animal were analysed for the presence of MN. The other six rats per group were subcutaneously implanted with osmotic pumps containing 5-bromo-2′-deoxyuridine (BrdUrd), three days prior to necropsy. Paraffin sections were made from the nasal cavity (four levels) of these animals for histopathology and cell-proliferation measurements. The frequency of cells with MN was not increased in any of the groups. However, there was also no induction of MN in nasal cells of rats exposed to a single dose of cyclophosphamide (CP, 20 mg/kg) by gavage and analysed 3, 7, 14 or 28 days after the treatment. In contrast, nasal epithelial cells from rats exposed to 10 or 15 ppm FA vapour showed clear site-specific pathological changes (focal epithelial degeneration, inflammation and squamous metaplasia) in a decreasing gradient (anterior to posterior). Analysis of slides after anti-BrdUrd antibody staining clearly indicated increased cell proliferation (unit length labelling indices, ULLI) after exposure to 6 ppm and higher. No treatment-related effects were measured after exposure to 0.5, 1 and 2 ppm. When comparing the cell-proliferation rate of normal epithelium with that of directly adjacent metaplastic epithelium, no consistent pattern was found: depending on the location, cell proliferation of normal epithelia was either higher or lower. Our results support previous findings demonstrating local cytotoxic effects in the nose of rats after inhalation of FA. However, induction of MN in the nasal epithelium as an indicator of a mutagenic effect was not seen. Because only limited experience exists for the MNT with rat nasal epithelial cells, this result has to be interpreted with great care. The contribution of mutagenicity to FA's carcinogenicity in rat nasal epithelium remains unclear.