The Effect of High-Dose Thiotepa, Alone Or in Combination with Other

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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, cytosine 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 day 0, each group received an ‘induction-like’ irradiation and/or cytotoxic-containing conditioning regimens (see text). Adoptive transfer to untreated BALB/c mice was performed 2–11 days from the conclusion of treatment, and the mice were followed for the appearance of leukemia without further manipulation. BCL1 ¼ B-cell leukemia/lymphoma.

100 100

80 80

60 60 Group 50 BU-CY BU-TH 40 TH-CY 40 Group Untreated 30 BU-CY BU-TH Leukemia free survival (%) Leukemia free survival (%) 20 20 TH-CY Untreated 10

0 0 0 10 20 30 40 50 60 70 80 90 100 110 20 40 60 80 100 Days Days

7 Figure 2 Time to development of leukemia following inoculation of Figure 3 Survival of BALB/c mice following inoculation with 10 BCL1 BALB/c mice with 107 leukemic cells, treated with BU–CY, BU–TH, followed by treatment with either BU–CY, BU–TH, TH–CY or untreated TH–CY, and the untreated leukemia control group followed by adoptive leukemia control group followed by adoptive transfer on day þ 11. transfer on day þ 5. Experiments were repeated twice (n ¼ 9 or 10). BU– Experiments were repeated twice (n ¼ 8 or 10). BCL1 ¼ B-cell leukemia/ CY ¼ busulfan–cyclophosphamide combination; BU–TH ¼ busulfan–thio- lymphoma; BU–CY ¼ busulfan–cyclophosphamide combination; BU– tepa combination; TH–CY ¼ thiotepa–cyclophosphamide combination. TH ¼ busulfan–thiotepa combination; TH–CY ¼ thiotepa–cyclophosphamide combination. treated with either 15 or 25 mg kgÀ1 of TH, and compared regimens proved to be the most effective ones (Figures 2 with leukemic control mice (with six mice per group). All and 3). CY–TH and BU–CY combinations had similar groups were monitored on the weekly basis for the results. Comparison of the effect of TH–CY treatment appearance of leukemic cells in the peripheral blood, or versus BU–TH showed only borderline signiﬁcance leukemia-related death. Time to onset of leukemia ranged (P ¼ 0.067) in favor of TH–CY, when AT was done on between 22 and 42 days in the untreated group. Onset of day 5. However, the BU–TH combination was found leukemia in animals treated with TH ranged from 20 to 55 ineffective when AT was performed on day þ 11 (Figure 3). and 27 to 49 days in the 15 and 25 mg kgÀ1 TH dose groups, respectively. Similarly, no difference was noted in the leukemia-related death. Thus, TH alone demonstrated no Thiotepa alone does not treat high leukemic load, but signiﬁcant antileukemic activity in the treatment set-up produces better synergism than TBI when combined with without AT. cyclophosphamide The next step was to evaluate the effect of TH with high tumor load. In this experiment, antileukemic treatment was Cyclophosphamide-containing regimen is extremely postponed for 7 days after inoculation with 107 spleen effective against BCL1, whereas thiotepa is not BCL1 cells. AT of 105 cells was administered 2and 8 days In the second experiment, treatment was started 24 h after treatment with TH, TH–CY or CY–TBI. The results after inoculation with 107 leukemic cells. AT of 105 cells were checked against leukemia control mice that were was performed 5 and 11 days later. The CY-containing inoculated with leukemia and received no further treat-

Bone Marrow Transplantation Thiotepa and lymphoid malignancies A Abdul-Hai et al 894 100 100

80 80 Group BU TH 60 60 Untreated

Group 40 CY TBI 40 TH TH CY Leukemia free survival (%) Untreated Leukemia free survival (%) 20 20

0 0 0 20 40 60 80 100 120 0 20 40 60 80 100 Days Days

Figure 4 Comparison of survival following treatment of BALB/c mice Figure 5 Comparative efﬁcacy of thiotepa, busulfan on BCL1 leukemia. with CY–TBI, TH or TH–CY, and leukemic control. In this study, high Experiments were repeated twice (n ¼ 9 or 10). BCL1 ¼ B-cell leukemia/ tumor load was achieved by inoculation with 107 spleen BCL1 cells and lymphoma. postponing of treatment till day þ 7. Synergism of TH with CY was better than the CY–TBI synergism. Experiments were performed twice (n ¼ 9or 10). BCL1 ¼ B-cell leukemia/lymphoma; CY–TBI ¼ cyclophosphamide– total-body irradiation; TH–CY ¼ thiotepa–cyclophosphamide combina- 100 tion.

80 ment. TH alone had no signiﬁcant effect as compared to the control group. However, a synergistic effect was seen in the combination of TH–CY: none of the animals developed leukemia, whereas 4/10 animals in the CY–TBI group did 60 (P ¼ 0.029, Figure 4).

Thiotepa demonstrates better performance against BCL1 40 leukemia compared with busulfan and etoposide Inoculation of 107 BCL1 was followed by high-dose Leukemia free survival (%) treatment with TH, BU, BU–CY or TH–CY. AT with 20 Group 106 spleen cells was administered on day þ 7. Results were TH then compared with the untreated leukemia control group. Untreated BU treatment did not produce a signiﬁcant effect against BCL1 (all animals developed leukemia after a median of 33 0 days). Following treatment with TH, in this experiment, 6/9 0 20 40 60 80 100 120 animals developed leukemia at a median of 41 days Days (P ¼ 0.003, Figure 5). Here, combining CY with either Figure 6 Inoculation of higher doses of BCL1 (108 cells) with or without drug was equally effective (0/7 and 0/10 animals developing sequential treatment with thiotepa, followed by adoptive transfer with 105 leukemia in the BU–CY and TH–CY combinations, spleen cells on day þ 6. BCL1 ¼ B-cell leukemia/lymphoma. respectively). Inoculation with higher doses of BCL1 (108 cells) was followed by high-dose treatment with TH, BU, etoposide (P ¼ 0.003, Figure 6). Here, combining either drug with CY (VP16), melphalan (MEL) or the TH–VP16, TH–MEL, was equally effective (0/7 and 0/10 animals developing BU–TH, BU–TH–MEL combinations. AT with 105 spleen leukemia in the BU–CY and TH–CY groups, respectively). cells was performed on day þ 6. Results were again Interestingly, in this model, the combination of BU with compared to the untreated leukemia control group. BU MEL was antagonistic. When administered alone, MEL treatment had no signiﬁcant effect against BCL1 (all eradicated BCL1 cells and none of the AT animals animals developed leukemia after a median of 33 days), developed leukemia. Likewise, when MEL was given in while following treatment with TH, in this experiment, combination with TH, none of the animals developed 6/9 animals developed leukemia at a median of 41 days leukemia. However, the TH–BU–MEL combination

Bone Marrow Transplantation Thiotepa and lymphoid malignancies A Abdul-Hai et al 895 Table 2 Thiotepa in multi-drug high-dose chemotherapy trans- study, only CY and MEL were more effective as single plant conditioning regimens agents within the same experimental framework. Protocol Leukemia induction I Leukemia induction II In spite of its disappointing performance as a single agent, TH efficacy was shown when used in conjunction Time to leukemia onset after adoptive transfer (in days) with other agents. A synergistic or additive effect of TH Leukemia control 35, 42 Â 5, 71, 498 Â 322,24Â 5, 35 when combined with other agents was established. The BECAM 498 Â 10 59, 481 Â 9 combination of TH and CY was as effective as the BETCAM 498 Â 10 481 Â 10 commonly used BU–CY combination, and more effective Abbreviations: A ¼ cytosine arabinoside; B ¼ BiCNu; C ¼ cyclophos- than the BU–TH combination. Additionally, we have phamide; E ¼ etoposide; M ¼ melphalan; T ¼ thiotepa. found that the combination of TH–CY was more effective The figures represent the time (number of days) until leukemia appeared. In than CY–TBI. These results are promising since TH may be cases where leukemia did not appear, time values represent the last day of burdened with fewer short- and long-term side effects than follow-up (marked with the ‘4’ sign). Leukemia induction I—inoculation of 106 BCL1 cells followed by adoptive transfer of 105 splenocytes on day BU (such as generalized or myoclonic seizures, veno- +7 into normal BALB/c mice. Leukemia induction II—inoculation of 107 occlusive disease (VOD) and a syndrome of diffuse BCL1 cells followed by adoptive transfer of 105 splenocytes on day +7. interstitial pulmonary fibrosis, persistent cough, fever, rales and dyspnea also known as ‘busulfan lung’15,16) or TBI yielded no antileukemic effect (all animals developed (with common side effects such as alterations in pulmonary leukemia, Po0.001). functions, formation of cataract or sicca syndrome, hypothyroidism and the development of hypogonadism and disturbed growth in children15,17) thus making TH an TH in multi-drug regimens excellent candidate for inclusion in pre-BMT myeloablative TH was added to known BMT-conditioning regimens and non-myeloablative conditioning protocols. Unfortu- commonly used in human malignant lymphoma. The nately, in the experimental set-up of our study and due to dosage of the chemotherapeutic agents and the adminis- the efficacy of other drugs, we have not been able to tration schedules were left unchanged. Based on the results examine thoroughly the efficacy of TH when incorporated presented in Table 2, we could not identify the role of TH with multi-drug conditioning protocols (Table 2). when incorporated with these regimens. However, one conclusive finding is the following: while combining BU with CY or with TH did not change the conditioning efficacy, the combination of BU and MEL Discussion may be antagonistic since the use of MEL alone or in combination with TH prevented totally the development of Thiotepa is an alkylating agent that reacts with DNA the experimental leukemia, while with the TH–BU–MEL phosphate groups to produce cross-linking of DNA strands combination, all animals developed leukemia (Po0.001). leading to inhibition of DNA, RNA and protein synthesis. We conclude that TH emerges as a good candidate for Although its mechanism of action has not been explored as pre-stem cell transplantation conditioning regimens in thoroughly as that of other alkylating agents, it is presumed lymphoid malignancies, and ought to be evaluated in that the aziridine rings open and react as nitrogen mustard; allogeneic murine models. This report should be followed reactivity is enhanced at a lower pH. Its common side with clinical studies; in addition, further evaluation is called effects include myelosuppression, dizziness, fever, headache for to assess the possible antagonistic interaction between and anorexia. Thiotepa is believed to carry a low emetic BU and melphalan. potential (o10%). It has been used clinically in several conditions, including breast adenocarcinoma,2 Hodgkin’s 5 14 disease, ovarian adenocarcinoma and transitional cell Acknowledgements carcinoma of the bladder.3,4 In the context of BMT, TH has been used for multiple myeloma,6 breast carcinoma,7 Dr Shapira’s work is supported by the Dr Sima Lior fund. leukemia8 and lymphoma9 due to its relative safety and a low-toxicity profile (other than the expected myelosuppression), which render TH preferable over other agents References when used in high-dose protocols, without causing further toxicity even in high-risk groups.6 Yet, the role of TH in 1 Shay H, Zarafonetis C, Smith N, Wolodow I, Sun DC. combination with other drugs in lymphatic diseases has not Treatment of leukemia with triethylene thiophosphoramide been sufficiently studied. (thio-TEPA); preliminary results in experimental and clinical In this report, we present a series of experiments designed leukemia. Arch Intern Med 1953; 92: 628–645. to investigate the efficacy of TH and its role in combination 2Ghalie R, Richman CM, Adler SS, Cobleigh MA, Korenblit with other cytotoxic agents in a murine lymphoid leukemia AD, Manson SD et al. Treatment of metastatic breast cancer model. We have found that TH cannot be used as a single with a split-course high-dose chemotherapy regimen and À1 autologous bone marrow transplantation. J Clin Oncol 1994; agent (even in very high doses—15–25 mg kg ) for treating 12: 342–346. experimental murine leukemia. However, it was more 3 Horn Y, Eidelman A, Walach N, Yuval E, Markowitz A. effective than several drugs commonly used in BMT Treatment of superficial bladder tumors in a controlled protocols such as BU and VP-16 (Figure 5), and produced trial with thio-tepa versus adriamycin. J Surg Oncol 1984; 27: better synergism with CY than TBI (Figure 4). In our 67–69.

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