Cyclophosphamide Alters the Gene Expression Profile in Patients Treated with High Doses Prior to Stem Cell Transplantation Ibrahim El-Serafi 1 , Manuchehr Abedi-Valugerdi 1 , Zuzana Pota ´ cova ´ 1,2 , Parvaneh Afsharian 1,3 , Jonas Mattsson 4,5 , Ali Moshfegh 6 , Moustapha Hassan 1,2 * 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 Abstract 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. Citation: El-Serafi I, Abedi-Valugerdi M, Pota ´cova ´ Z, Afsharian P, Mattsson J, 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 Editor: Antonio Perez-Martinez, Hospital Infantil Universitario Nin ˜o Jesu ´ s, Spain Received November 6, 2013; Accepted December 15, 2013; Published January 22, 2014 Copyright: ß 2014 El Serafi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 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. * E-mail: moustapha.hassan@ki.se Introduction 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 non- malignant diseases such as metabolic and haematological disor- ders. 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 adminis- tered 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 cross- linking. 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 PLOS ONE | www.plosone.org 1 January 2014 | Volume 9 | Issue 1 | e86619