Cyclophosphamide Alters the Gene Expression Profile in Patients Treated with High Doses Prior to Stem Cell Transplantation

et al. (2014) Cyclophosphamide Alters the Gene Expression Profile in Patients Treated with High Doses Prior to Stem Cell Transplantation. PLoS ONE 9(1): e86619. doi:10.1371/journal.pone.0086619 Cyclophosphamide Alters the Gene Expression Profile in Patients Treated with High Doses Prior to Stem Cell Transplantation Ibrahim El-Serafi 0 Manuchehr Abedi-Valugerdi 0 Zuzana Pota cova 0 Parvaneh Afsharian 0 Jonas Mattsson 0 Ali Moshfegh 0 Moustapha Hassan 0 Antonio Perez-Martinez, Hospital Infantil Universitario Nin o Jesu s, Spain 0 1 Experimental Cancer Medicine (ECM), Clinical Research Centre (KFC), Department of Laboratory Medicine, Karolinska Institutet, Huddinge, Stockholm, Sweden, 2 Clinical Research Centre (Novum), Karolinska University Hospital-Huddinge, Stockholm, Sweden, 3 Department of Genetics, Royan Institute , Tehran , Iran , 4 Centre for Allogeneic Stem Cell Transplantation, Karolinska University Hospital-Huddinge, Stockholm, Sweden, 5 Department of Therapeutic Immunology, Karolinska Institute, Karolinska University Hospital, Huddinge, Stockholm, Sweden, 6 Cancer Centre of Karolinska (CCK), Department of Oncology-Pathology, Karolinska Institutet , Solna, Stockholm , Sweden Background: Hematopoietic stem cell transplantation is a curative treatment for several haematological malignancies. However, treatment related morbidity and mortality still is a limiting factor. Cyclophosphamide is widely used in condition regimens either in combination with other chemotherapy or with total body irradiation. Methods: We present the gene expression profile during cyclophosphamide treatment in 11 patients conditioned with cyclophosphamide for 2 days followed by total body irradiation prior to hematopoietic stem cell transplantation. 299 genes were identified as specific for cyclophosphamide treatment and were arranged into 4 clusters highly down-regulated genes, highly up-regulated genes, early up-regulated but later normalized genes and moderately up-regulated genes. Results: Cyclophosphamide treatment down-regulated expression of several genes mapped to immune/autoimmune activation and graft rejection including CD3, CD28, CTLA4, MHC II, PRF1, GZMB and IL-2R, and up-regulated immune-related receptor genes, e.g. IL1R2, IL18R1, and FLT3. Moreover, a high and significant expression of ANGPTL1 and c-JUN genes was observed independent of cyclophosphamide treatment. Conclusion: This is the first investigation to provide significant information about alterations in gene expression following cyclophosphamide treatment that may increase our understanding of the cyclophosphamide mechanism of action and hence, in part, avoid its toxicity. Furthermore, ANGPTL1 remained highly expressed throughout the treatment and, in contrast to several other alkylating agents, cyclophosphamide did not influence c-JUN expression. - Funding: The authors wish to acknowledge the Swedish Cancer Society and the Swedish Childhood Cancer Foundation for their support. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Hematopoietic stem cell transplantation (HSCT) is currently used as a curative treatment for a wide range of diseases, including malignancies such as leukaemia and lymphomas and nonmalignant diseases such as metabolic and haematological disorders. Conditioning regimens - either a combination of cytostatics or cytostatics together with radiotherapy - are designed to prepare the patient to receive donor stem cells. Cytostatics are administered in order to eliminate malignant cells, provide free space for the donor cells and suppress the immune system in order to prevent graft rejection [1]. Cyclophosphamide (Cy) is an alkylating agent widely used as a part of the conditioning regimen prior to HSCT and for the treatment of haematological malignancies and solid tumours. Cy is used either in combination with other cytostatics or with total body irradiation (TBI) [2]. It acts on DNA by attaching an alkyl group to the guanine base of DNA at the number 7 nitrogen atom of the imidazole ring and leads to guanine-adenine intra-strand crosslinking. This damage to the DNA strand can trigger apoptosis when the cellular machinery fails to repair it. Cy is also a potent immunosuppressive agent i.e. it is capable of attenuating both humoral and cell-mediated immune responses [3]. Due to these immunosuppressive effects, Cy is used in the treatment of several autoimmune diseases including rheumatoid arthritis and systemic lupus erythematosus [4]. Cy is a prodrug that is metabolized by cytochrome P450 to its main active metabolite, 4-hydroxycyclophosphamide (4-OH-Cy), which comprises about 90% of the total Cy dose [5]. 4-OH-Cy is subsequently converted to phosphoramide mustard and acrolein, which is nephrotoxic. An alternative pathway of Cy transformation is N-dechloroethylation. Moreover, Cy is partly metabolized to the inactive metabolite 2-dechloroethyl-Cy and to chloroacetaldehyde, which is neurotoxic [68]. Studies on Cy kinetics have shown a high inter-individual variation in elimination half-life and clearance [6,9]. This variation may be explained by polymorphisms in CYP2B6, the main enzyme responsible for the conversion of Cy to its active form [1012]. Several studies have investigated the clinical efficacy of Cy alone or in combination with other cytostatics or radiotherapy [2,13]. However, the contribution of Cy to the outcome of HSCT and, more importantly, the mechanisms by which Cy exerts its effect on immune cells has not yet been addressed. In the past decade, the advent of DNA microarray technology together with the availability of the complete nucleotide sequence of the human genome have allowed elucidation of the molecular mechanisms in several diseases [14,15] or treatment regimens [16,17]. In order to understand the effect of high dose cyclophosphamide on different genes, we employed the DNA microarray to investigate the gene expression profile in peripheral mononuclear cells of patients suffering from haematological malignancies and undergoing conditioning regimen consisting of Cy followed by TBI. We followed the gene profile pre-, during and after treatment. Materials and Methods Patients and Treatment Eleven patients were enrolled in this study. The patients were admitted at the Centre for Allogeneic Stem Cell Transplantation (CAST), Karolinska University Hospital-Huddinge. The study was approved by the ethical committee of Karolinska Institutet (616/ 03) and written informed consent was obtained from the patients or, in case of paediatric patients, their parents. Twelve samples from healthy donors, who had given their written consent, were run concomitantly as negative controls. The ethics committee approved the consent procedure. Six of these patients were diagnosed with acute lymphocytic leukaemia (ALL), two with acute (...truncated)