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© 1999 American Society for Clinical Oncology Successful Treatment of Metastatic Renal Cell Carcinoma With a Nonmyeloablative Allogeneic Peripheral-Blood Progenitor-Cell Transplant: Evidence for a Graft-Versus-Tumor EffectFrom the Bone Marrow Transplant Unit, Hematology Branch, National Heart, Lung and Blood Institute, Bethesda, MD. Address reprint requests to Richard W. Childs, MD, Hematology Branch, Building 10, Room 7C103, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892; email childsr{at}nih.gov
PURPOSE: A 50-year-old man developed progressive pulmonary metastasis resistant to interferon alfa-2b treatment 7 months after he underwent left nephrectomy for stage III renal cell carcinoma. We performed a nonmyeloablative allogeneic peripheral-blood stem-cell transplant in this patient to exploit a possible graft-versus-tumor effect from allogeneic lymphocytes. MATERIALS AND METHODS: The conditioning regimen consisted of fludarabine and cyclophosphamide followed by a T-cell replete, granulocyte-colony stimulating-factor–mobilized peripheral-blood stem-cell transplant from his HLA-identical brother. Cyclosporine was administered from days –4 to +45 to prevent graft rejection and acute graft-versus-host disease (GVHD). RESULTS: Serial polymerase chain reaction analysis of hematopoietic lineage-specific minisatellites initiallyshowed mixed chimerism in CD14+ and CD15+ myeloid cells, CD3+ T cells, and CD34+ progenitor cells, with rapid conversion to 100% donor T-cell chimerism by day +60 and 100% donor myeloid cells by day +100. Serial computed tomography scans of the chest showed stable disease at day +30, slight regression of pulmonary lesions at day +63, and complete disappearance of all pulmonary metastatic disease by day +110. Mild transient acute GVHD disease of the skin occurred on day +60 and limited chronic GVHD of the skin occurred by day +200. CONCLUSION: The complete regression of metastatic disease, which has now been maintained for more than 1 year, is compatible with a graft-versus-tumor effect.
FEW ADVANCES HAVE BEEN made in the treatment of metastatic renal cell carcinoma (RCC). The disease is largely resistant to hormonal and cytotoxic chemotherapy,1 but approximately 20% of patients respond to interleukin-2 or interferons. However, treatment-related toxicity can be substantial, and there is no evidence that such immunomodulators prolong survival.2,3 It is now clear that after allogeneic bone marrow transplantation, donor T cells can exert a powerful graft-versus-tumor (GVT) effect in myeloid and lymphoid leukemias, lymphoma, multiple myeloma, and possibly breast cancer.4-8 There is some evidence in RCC that autologous T lymphocytes exert an immune antitumor response: Tumor-infiltrating lymphocytes can be readily isolated from some RCCs and have been shown in vitro to have HLA-restricted specific cytotoxicity against tumor cells.9 Because RCC has been shown in vitro to be a target for T-cell cytotoxicity and potential renal tissue-restricted minor histocompatibility antigens have been described,10 we postulated that a GVT effect might be generated after transplantation of alloreactive lymphocytes that recognize tumor-specific or recipient-specific alloantigens. We therefore investigated the use of immunosuppressive but nonmyeloablative, low-dose preparative regimens and peripheral-blood stem-cell allotransplants ("minitransplants") to achieve rapid engraftment of donor T cells and to exploit a GVT effect in patients with metastatic RCC. Such nonmyeloablative transplants have the advantage of low transplant-related mortality with the establishment of full donor lymphoid chimerism.11,12 The patient described here is the first to be treated on this investigational protocol and had complete resolution of progressive and extensive metastatic disease after this novel transplant approach.
Evaluation of Chimerism After Transplantation DNA extracts from peripheral-blood mononuclear cells were analyzed by polymerase chain reaction (PCR) to identify differences in minisatellite regions between donor and recipient. After transplantation, serial samples of blood and marrow were analyzed for donor-recipient chimerism using informative PCR primers. Chimerism in different cell lineages was measured in the following way. First, RBCs were lysed, then myeloid cells were isolated by positive selection using anti-CD14– and anti-CD15–coated immunomagnetic beads. T cells were isolated from the residual (CD14-/CD15-) sample using anti-CD3–coated immunomagnetic beads. Approximately 30 mL bone marrow aspirate was obtained on days +30, +60, and +100 after transplantation and was additionally sorted for B cells, erythroid, and CD34+ progenitors using immunomagnetic beads coated with monoclonal antibodies for CD19, CD71, and CD34, respectively. DNA was extracted from selected cells and suspended in Tris-acetate and EDTA at a concentration of 100 ng/µL. Samples were stored at 4°C until the time of analysis. PCR primers flanking the repeat units of the minisatellite YNZ-22 were used to amplify this informative sequence in the donor and patient before and after transplantation according to a previously published method.13 In addition, dilutions of pretransplant patient and donor DNA were used as controls to establish in a semiquantitative fashion the proportion of donor chimerism in the posttransplant samples. PCR products were electrophoresed in a 1.5% agarose gel at 60 V for 4 hours. The product was then transferred to a nylon membrane and probed with a YNZ-22 minisatellite specific oligonucleotide.
Case History The patient remained well without evidence of recurrent disease until September 1997, when he presented with a persistent, nonproductive cough and weight loss. A CT scan of the chest showed more than 40 nodules scattered throughout both lung fields, consistent with diffuse pulmonary metastatic disease. CT scans of the abdomen and pelvis were normal, with the exception of a small filling defect in the inferior vena cava, considered to be either postoperative changes or persisting tumor thrombus. He was treated with oral megestol acetate and a 10-week course of subcutaneous interferon alfa-2b 3 MU/m2 three times per week. Repeat CT scans of the chest in December 1997 showed an increase in the size of most of the lung nodules and new nodules, consistent with disease progression. In view of the low probability of response to further conventional treatment for metastatic RCC, he was referred to The National Institutes of Health for experimental treatment. He received an institutionally approved investigational protocol (NIH #97H-00196) designated to evaluate GVT effects after nonmyeloablative allogeneic transplantation for RCC. Patients with nonresectable, biopsy-proven, metastatic RCC that progressed despite immunomodulatory therapy, with or without chemotherapy, are eligible for this protocol. Before transplantation, a fine-needle aspirate of a pulmonary nodule confirmed metastatic RCC (Fig 1). A brain magnetic resonance imaging scan was negative for metastatic disease. A pretransplant baseline CT scan again showed an increase in the size of most of the previously observed pulmonary nodules as well as multiple new lung nodules, consistent with ongoing and rapid disease progression. Approximately 12 weeks after the discontinuation of interferon alfa-2b therapy, the patient was prepared for transplantation with cyclophosphamide 60 mg/kg intravenously on 2 successive days followed by fludarabine 25 mg/m2 intravenously daily for 5 days. Cyclosporine was started on day –4 to prevent graft rejection and as prophylaxis for graft-versus-host disease (GVHD). His HLA-identical brother (HLA type: A29,8,001; B40,45; DRß1*11,15) was given granulocyte colony-stimulating factor 10 µg/kg for 5 days, and a T-cell replete peripheral-blood stem-cell allograft was collected by leukapheresis on the fifth day of granulocyte colony-stimulating factor administration. On day 0, the patient received a transplant of 9.1 x 106 CD34+ cells/kg and 4.2 x 108 CD3+ T cells/kg from his brother. Pancytopenia was brief, with only 10 days of neutropenia and a platelet nadir of 40 x 109/L, and did not require platelet transfusion. His posttransplant course was uncomplicated with the exception of a perianal fistula that was surgically closed as an outpatient on day +40. He was discharged to outpatient follow-up on posttransplant day +14. Cyclosporine was tapered and stopped on day +45 after a chest CT on day +30 showed no change in the metastatic pulmonary disease. Transient grade 1 GVHD of the skin occurred on day +60 but resolved rapidly without treatment. Subsequent CT scans on days +63, +86, and +110 showed regression and ultimate resolution of all pulmonary metastatic disease (Fig 2). On day +102, he developed persistent symptoms of gastrointestinal reflux disease, and, after endoscopic biopsy, he was found to have cytomegalovirus esophagitis and gastritis that was successfully treated with intravenous ganciclovir. On day 200, limited chronic GVHD of the skin developed but improved rapidly after treatment with prednisolone 20 mg on alternate days and the reinstitution of oral cyclosporine. His immunosuppressive treatment for nearly resolved chronic skin GVHD was being tapered at the time of this writing, and he remained free of metastatic RCC 15 months after transplantation.
Chimerism Analysis
Every year in the United States alone, approximately 12,000 individuals die from advanced RCC.1 Most patients with metastatic disease rapidly progress, with median survival times between 12 and 16 months.2 Renal cell tumors are notoriously resistant to hormonal and cytotoxic chemotherapeutic agents, and there is no consensus on the best treatment approach for metastatic disease. The most active agents show response rates between 6% and 20% and do not prolong survival.14 Eight to twenty percent of patients can respond to immunomodulators such as interferon alfa-2b or gamma and interleukin-2, but durable and complete responses occur in less than 4% of patients.1,15,16 A recent phase III trial that compared interferon gamma-1b with placebo showed no difference in response rates, time to disease progression, or survival between groups.3 Another randomized study found that a combination of interleukin-2 and interferon alfa-2b achieved an 18.6% response in metastatic RCC but without survival benefit.15 These recent studies highlight the inadequacy of current therapy for metastatic RCC. However, there is strong evidence that RCC is susceptible to T-cell–mediated cytotoxicity: Tumor-infiltrating lymphocytes isolated from some renal cell tumors and T-cell clones isolated from some patients with RCC have been shown to have HLA-restricted specific cytotoxicity against renal tumor cells.9,17 Patients with advanced metastatic RCC sometimes undergo spontaneous disease regression, implicating an autologous antitumor immune response. However, to date, only a few RCC tumor antigens have been characterized.17,18 Because RCC seems to be susceptible to immune-mediated control, we explored a possible role of alloreactive lymphocytes to treat RCC refractory to conventional management. It is not known if allogeneic stem-cell transplants can confer a GVT effect in RCC. Recipient-specific minor histocompatibility antigens are possible targets for a GVT effect of donor cytotoxic T cells. Minor antigens that are present ubiquitously in many tissues could induce both GVT effects and GVHD; however, some antigens are tissue-restricted and may induce a more restricted alloresponse to tumor tissue.19,20 There is evidence for such tissue-restricted minor histocompatibility antigens on renal cells, which, if present on malignant renal cells, could serve as targets for donor T cells.10 Because RCC is typically refractory to most chemotherapeutic agents and does not respond to dose-intensification, we used a nonmyeloablative conditioning regimen that provided enough immunosuppression to allow engraftment of donor cells without causing the substantial side effects of conventional high-dose myeloablative preparative regimens. Fludarabine-based conditioning regimens are effective in providing adequate donor cell engraftment with minimum toxicity.11,12 In this patient and in 16 patients with other diseases, we observed a high rate of engraftment with low toxicity using this fludarabine and cyclophosphamide regimen. We attribute the complete regression of biopsy-proven and radiographically progressive pulmonary renal metastasis in this patient to a GVT effect for the following reasons. First, the chemotherapeutic agents used have no known activity in metastatic RCC. Indeed, CT scans performed 30 days after transplantation showed no evidence of tumor regression. Second, the 2-month interval between the transplant chemotherapy and the first signs of tumor regression is more compatible with an immune-mediated GVT effect. A 2- to 6-month time period is usually required for a graft-versus-leukemia effect from donor lymphocyte transfusions used to treat relapsed leukemia after allogeneic marrow transplantation.21 Furthermore, tumor regression did not begin until full donor T-cell chimerism was established. Although RCC can occasionally show spontaneous regression, the presence of 100% donor lymphoid chimerism indicates that if regression were immunologically induced, the principal effector cells were of donor origin. It is unclear whether recipient antigen-presenting cells, present during the initial state of mixed chimerism, contributed to or were necessary for this antitumor effect. Finally, tumor regression occurred shortly after the withdrawal of cyclosporine and the occurrence of mild acute GVHD of the skin. Graft-versus-leukemia effects have also been observed in conjunction with cyclosporine reduction and the occurrence of acute GVHD.22 The patient reported here is the first to be treated on this investigational protocol evaluating GVT effects in patients with metastatic RCC. Although this patient had a dramatic and complete response to this new treatment approach, further allogeneic transplants in similar patients are required to determine the place of nonmyeloablative allogeneic stem-cell transplantation in the treatment of metastatic RCC.
1. Savage PD: Renal cell carcinoma. Curr Opin Oncol 8:247-251, 1996[Medline] 2. Elson PJ, Witte RS, Trump DI: Prognostic factors for survival in patients with recurrent or metastatic renal cell carcinoma. Cancer Res 48:7310-7313, 1988[Medline]
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Gleave ME, Elhilali M, Fradet Y, et al: Interferon gamma–1b compared with placebo in metastatic renal-cell carcinoma. N Engl J Med 338:1265-1271, 1998 4. Weiden P, Flournoy N, Thomas E, et al: Antileukemic effects of graft-versus-host disease in human recipients of allogeneic marrow grafts. N Engl J Med 300:1068-1073, 1979[Abstract]
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Horowitz M, Gale RP, Sondel P, et al: Graft-versus-leukemia reactions after bone marrow transplantation. Blood 78:2120-2130, 1991 6. van Besien KW, de Lima M, Giralt SA, et al: Management of lymphoma recurrence after allogeneic transplantation: The relevance of graft-versus-lymphoma effect. Bone Marrow Transplant 19:977-982, 1997[Medline]
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Eibl B, Schwaighofer H, Nachbaur D, et al: Evidence for a graft-versus-tumor effect in a patient treated with marrow ablative chemotherapy and allogeneic bone marrow transplantation for breast cancer. Blood 88:1501-1508, 1996 8. Verdonck L, Lokhorst H, Dekker A, et al: Graft-versus-myeloma effect in two cases. Lancet 347:800-801, 1996[Medline] 9. Caignard A, Guillard M, Gaudin C, et al: In situ demonstration of renal-cell carcinoma specific T-cell clones. Int J Cancer 66:4:564-570, 1996 10. Poindexter N, Shenoy S, Howard T, et al: Allograft infiltrating cytotoxic T lymphocytes recognize kidney-specific human minor histocompatibility antigens. Clin Transplant 11:174-177, 1997[Medline]
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Slavin S, Nagler A, Naparstek E, et al: Nonmyeloablative stem cell transplantation and cell therapy as an alternative to conventional bone marrow transplantation with lethal cytoreduction for the treatment of malignant and nonmalignant hematologic diseases. Blood 91:756-763, 1997
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Childs R, Clave E, Barrett J, et al: Kinetics of engraftment in non-myeloablative allogeneic peripheral blood stem cell transplants: An analysis of hematopoetic-lineage chimerism. Blood 92:520, 1998 (abstr 2137) 13. Mackinnon S, Barnett L, Bourhis JH, et al: Myeloid and lymphoid chimerism after T-cell depleted bone marrow transplantation: Evaluation of conditioning regimens using the polymerase chain reaction to amplify human minisatellite regions of genomic DNA. Blood 180:3235-3241, 1992 14. Yagoda A, Abi-Rached B, Petrylak D, et al: Chemotherapy for advanced renal cell carcinoma: 1983-1993. Semin Oncol 22:42-60, 1995[Medline]
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Negrier S, Escudier B, Lassett C, et al: Recombinant human interleukin-2, recombinant human interferon alfa-2a, or both in metastatic renal-cell carcinoma. N Engl J Med 338:1272-1278, 1998 16. Rosenburg SA, Yang JC, Topalian SL, et al: Treatment of 283 consecutive patients with metastatic melanoma or renal cell cancer using high-dose bolus interleukin 2. JAMA 271:907-913, 1994[Abstract] 17. Brouwenstijn N, Hoogstraten C, Verdegaal E, et al: Definition of unique and shared T-cell defined tumor antigens in human renal cell carcinoma. J Immunol 21:427-434, 1998 18. Gaugler B, Brouwenstijn N, Vantomme V, et al: A new gene coding for an antigen recognized by autologous CTL on a human renal cell carcinoma. Immunogenetics 44:323-330, 1996[Medline]
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Warren EH, Greenberg PD, Riddell SR: Cytotoxic T-lymphocyte-defined human minor histocompatibility antigens with a restricted tissue distribution. Blood 91:2197-2207, 1998
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van der Harst D, Goulmy E, Falkenburg F, et al: Recognition of minor histocompatibility antigens on lymphocytic and myeloid leukemic cells by cytotoxic T-cell clones. Blood 83:1060-1066, 1994 21. Barrett AJ, Malkovska V: Graft-versus-leukemia: Understanding and using the alloimmune response to treat haematological malignancies. Br J Haem 93:754-761, 1996[Medline] 22. Collins RH, Rogers ZR, Bennett M, et al: Hematologic relapse of chronic myelogenous leukemia following allogeneic bone marrow transplantation: Apparent graft-versus-leukemia effect following abrupt discontinuation of immunosuppression. Bone Marrow Transplant 10:391-395, 1992[Medline] Submitted October 30, 1998; accepted February 26, 1999. This article has been cited by other articles:
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Copyright © 1999 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
