The Effect of High-Dose Thiotepa, Alone Or in Combination with Other
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Bone Marrow Transplantation (2007) 40, 891–896 & 2007 Nature Publishing Group All rights reserved 0268-3369/07 $30.00 www.nature.com/bmt ORIGINAL ARTICLE The effect of high-dose thiotepa, alone or in combination with other chemotherapeutic agents, on a murine B-cell leukemia model simulating autologous stem cell transplantation A Abdul-Hai, L Weiss, D Ergas, IB Resnick, S Slavin and MY Shapira Department of Bone Marrow Transplantation and Cancer Immunotherapy, Hadassah–Hebrew University Medical Center, Jerusalem, Israel The use of thiotepa (TH) is increasing, especially in stem Introduction cell transplantation, mainly due to its safety and blood– brain barrier penetration. We evaluated the use of TH in a Thiotepa (TH, triethylene thiophosphoramide), an ethylene murine model simulating autologous stem cell transplan- amide, developed by Lederle Laboratories in 1952, tation, with or without additional agents. Between 1 and possesses mechlorethamine-like alkylating activity and 11 days following inoculation of BALB/c mice with 105– has been used clinically for over 35 years.1 It is of particular 108 B-cell leukemia (BCL1) cells (simulating pre-trans- use in breast cancer, mainly as second-line treatment.2 plant leukemia loads), each group received an ‘induction- TH has been given intrathecally for carcinomatous like’ irradiation and/or cytotoxic regimen. Animals were meningitis, and intravesically for bladder carcinoma.3,4 either followed without treatment, or an adoptive transfer Mild-to-moderate activity was observed in several solid (AT) was performed to untreated BALB/c mice. Admi- tumors and hematological malignancies.1,5 There has nistered alone without AT, high-dose TH did not change been, however, a sustained interest in defining clinical roles the time to appearance of leukemia. Nevertheless, in the for TH. AT experiments, TH as a single agent showed better In recent years, the use of the drug has been increasing in antileukemic activity than busulfan (BU). Cyclophos- view of the possibility to increase its dose significantly (by a phamide (CY)-containing regimens were the most factor of 10–22, in contrast with only a 3- to 5-fold increase effective, and the TH–CY combination was as effective in most of the other antitumor agents), accompanied with as the commonly used BU–CY combination, and more rescue by re-infusion of cryopreserved autologous bone effective than the BU–TH combination. Moreover, a marrow or peripheral stem cells.6–9 Thus, TH has become synergistic effect was seen in the TH–CY combination one of the most useful drugs in bone marrow transplant- (none of the animals developed leukemia, whereas 4/10 ation protocols. Yet no studies compared the efficacy of animals in the CY–TBI group developed leukemia high-dose TH in combination with other chemotherapeutic (P ¼ 0.029)). In conclusion, although TH produced only agents. a moderate effect against BCL1 leukemia when used Spontaneous murine B-cell leukemia of BALB/c alone, its combination with CY is promising and should be mice10 provides a high-quality experimental model for tested further in allogeneic murine models and clinical studying the effect of chemotherapy, with or without studies. radiotherapy, on a B-lymphoid neoplasm resembling Bone Marrow Transplantation (2007) 40, 891–896; human disease. doi:10.1038/sj.bmt.1705838; published online 3 September 2007 Our goal in the present series of experiments using the Keywords: minimal residual disease; B-cell leukemia/ described murine leukemia model was to evaluate the lymphoma (BCL1); total-body irradiation (TBI); thiotepa; efficacy of various doses of TH, and determine its role in cyclophosphamide combination with other drugs. Materials and methods Animals Two- to 4-month-old inbred male and female BALB/c mice Correspondence: Dr MY Shapira, Department of Bone Marrow were purchased from the Hebrew University School of Transplantation and Cancer Immunotherapy, Hadassah–Hebrew Uni- Medicine (Jerusalem, Israel) for use as leukemia recipients. versity Medical Center, PO Box 12000, Jerusalem 91120, Israel. E-mail: [email protected] Mice were kept under clean (SPF) conditions with Received 23 February 2007; revised 18 June 2007; accepted 23 July 2007; autoclaved cages and sawdust. Food and acidified water published online 3 September 2007 were supplied as required. Thiotepa and lymphoid malignancies A Abdul-Hai et al 892 Total-body irradiation (AT) in the different experiments is to differentiate between Recipient mice were placed in irradiation chambers on day disease stages that range from minimal residual disease À1 and exposed to a myeloablative dose of total-body (in which longer time to AT is needed to identify post- irradiation (TBI) with 200 cGy given twice daily on 3 induction residual disease) to high disease load (where a consecutive days at 56 cGy minÀ1 (900 cGy is lethal in this shorter time is needed). Untreated BALB/c mice receiving model if no additional spleen or marrow stem cells are 105 spleen cells obtained from each experimental group administered; data not shown). TBI was delivered by a were used to determine whether or not at the time of AT, Philips X-ray unit (250 kV, 20 mA) (Varian Oncology clonogenic BCL1 were still present in the spleen of treated Systems, Zug, Switzerland) with a 0.2-mm Cu filter. Source mice. Recipients were observed for the development of to skin distance was 70 cm. leukemia. Splenomegaly and peripheral blood lymphocyte counts were monitored to confirm the presence of leukemia, Chemotherapy and to determine whether leukemia was indeed the cause The chemotherapeutic agents used in this study included of death. TH, cyclophosphamide, busulfan (BU), melphalan, cyto- sine arabinoside, carmustine (BiCNu) or etoposide. The Statistical analysis dose chosen for each agent was calculated on a per Significance was determined using the Student’s t-test kilogram basis to yield doses comparable to, or higher and Kaplan–Meier survival analysis (medcalc, Belgium); than those used in clinical practice, particularly in BMT Pp0.05 was considered statistically significant. Studies protocols (Table 1). All chemotherapeutic agents and TBI were repeated 2–3 times. were administered on the same days. Murine B-cell leukemia Results B-cell leukemia/lymphoma (BCL1) was maintained in vivo in BALB/c mice by intravenous passage of 106–107 Experimental design peripheral blood lymphocytes obtained from tumor-bear- One day following inoculation of BALB/c mice with 105– ing mice. It has been shown previously that even a small 108 BCL1 leukemia cells (simulating various pre-transplant inoculum of BCL1 might be sufficient to cause typical leukemia loads), each group of mice received one of the leukemia in all recipients.11 All recipients of 410–100 ‘induction-like’ irradiation, and/or cytotoxic-containing BCL1 cells develop splenomegaly and marked lympho- conditioning regimens representing BMT induction meth- cytosis in the blood. Weekly peripheral blood lymphocyte ods. In several experiments, the mice were followed up for counts were carried out in all experimental animals. the appearance of leukemia without further manipulation. Due to the apprehension about early toxic death preventing Preparation of spleen cells for inoculation of BCL1 us from analyzing the antileukemic effect, and to evaluate lymphoma/leukemia and for adoptive transfer the effect of the induction treatment and the residual Spleens were removed aseptically from BALB/c mice disease, AT was performed for untreated BALB/c mice following leukemic inoculation and antileukemic treatment, 2–11 days from the conclusion of the treatment (Figure 1). then teased through a nylon mesh into RPMI 1640 medium The frame of reference for the various groups was the (Life Technologies, Grand Island, NY, USA) to create a results of most commonly used myeloablative conditioning single-cell suspension that was injected into the lateral tail regimens, for example high-dose cyclophosphamide and vein of each recipient. TBI (CY–TBI),12 or high-dose BU and cyclophosphamide (BU–CY).13 Adoptive transfer experiments assessing efficacy of the induction treatment on BCL1 Thiotepa by itself is not effective against BCL1 leukemia in To investigate minimal residual disease and to exclude early non-adoptive transfer studies toxic deaths, spleen cells from the treated mice were In the first experiment, we explored the effect of two very transferred to normal BALB/c mice at a constant timing. high TH doses and their antileukemic activity without AT. The purpose of variability in the timing of adoptive transfer Mice were inoculated with 107 BCL1 cells, then 24 h later Table 1 Cytotoxic drug doses and mode of administration Drug Dose (mg kgÀ1) Mode of application Supplier Thiotepa 15–25 i.v. or i.m. Bedford laboratories, USA Cyclophosphamide 120 i.v. Taro, Haifa, Israel Busulfan 16 p.o. Burroughs Wellcome, UK Melphalan 4 i.p. Burroughs Wellcome, UK Cytosine arabinoside 6.5 i.p. Upjohn, Kalnnazoo, MI, USA Carmustine (BiCNu) 8 i.p. Bristol-Myers Squibb, NJ, USA Etoposide 6.5 Â 3 i.p. Abic, Natanya, Israel Abbreviations: i.m. ¼ intramuscular; i.p. ¼ intraperitoneal; i.v. ¼ intravenous; p.o. ¼ per os. Bone Marrow Transplantation Thiotepa and lymphoid malignancies A Abdul-Hai et al 893 –1d, BCL1 d 0, induction Adoptive transfer 2-11 days Follow up for leukemia development inoculation treatment Figure 1 Graphical description of the protocol. On day À1, BALB/c mice were inoculated with BCL1 leukemia cells (105–108 cells simulating various pre- transplant leukemia loads). On