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Complete remission in a patient with recurrent acute myeloid leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity Leukemia (2004) 18, 165–166. doi:10.1038/sj.leu.2403186 Published online 6 November 2003 TO THE EDITOR
The transcription factor Wilms’ tumor protein 1 (WT1) has recently attracted much interest as target for cancer immunotherapy as reviewed by Rosenfeld et al1 in the July issue of Leukemia. It is strongly overexpressed in leukemic blasts in the majority of patients with acute myeloid leukemia (AML)2 and chronic myeloid leukemia as well as various carcinomas. WT1 plays a key role in AML, as its inhibition by antisense oligonucleotides leads to growth arrest, differentiation and apoptosis.3 HLA-A0201and HLA-A24-restricted epitopes from WT1 were identified and cytotoxic T cells raised against these epitopes lysed leukemic blasts but not hematopoietic stem cells.4 In animal models, a vigorous T-cell response can be elicited by vaccination with the WT1 epitope 126–134, which is homologous to the human epitope, resulting in rejection of WT1-expressing tumor cells.5 A phase I/II study has been initiated in our institution to analyze the immunogenicity and toxicity of WT1 126–134 peptide vaccination in AML patients. We report on the remarkable clinical course of the first patient with recurrent AML who achieved complete remission in the absence of significant toxicity. The patient was diagnosed with AML FAB M4 with a normal karyotype in June 2000. This 67-year-old woman initially achieved a complete remission after chemotherapy with cytarabine and mitoxantrone, which lasted for 16 months. Following relapse in December 2001 with 80% leukemic blasts in bone marrow, she was treated with one cycle of cytarabine and liposomal daunorubicin. The phase of myelosuppression was complicated by severe pulmonary aspergillosis, prompting cessation of further chemotherapy. The first bone marrow aspiration 4 weeks after completion of the chemotherapy when neutrophils had recovered to 1.9/nl but platelets were still low, showed less than 5% blasts. A partial remission was diagnosed 9 weeks later when peripheral blood cells had normalized and the proportion of blasts in bone marrow was 5–10% as illustrated in Figure 1. The patient was HLA-A201 positive and the first patient included in a WT1 vaccination protocol receiving four biweekly vaccinations with the HLA-A0201-binding WT1 peptide 126– 134 (RMFPNAPYL,4 Clinalfa, Switzerland) in a dose of 0.2 mg admixed with 1 mg of keyhole limpet hemocyanin (KLH, Immucothels, biosyn, Germany) as adjuvant injected i.d. and s.c. on day 0. GM-CSF (Leukomaxs, Essex Pharma, Germany) in a dose of 75 mg per day was injected s.c. at the same site (proximal thigh) as the WT1 peptide on days –2 to þ 1. The WT1 vaccination protocol was approved by the Institutional Ethics Committee. The combination of both adjuvants was chosen due to the immunological efficacy in melanoma peptide vaccination.6 While the patient had normal complete blood counts when vaccination was started, 2 weeks after the first Correspondence: Professor Dr U Keilholz, Charite´ – Universita¨tsmedizin Berlin, Campus Benjamin-Franklin, Medizinische Klinik III, Ha¨matologie, Onkologie und Transfusionsmedizin, Hindenburgdamm 30, Berlin 12200, Germany; Fax: þ 49-30-8445-4468; E-mail:
[email protected] Received 28 July 2003; accepted 10 September 2003; Published online 6 November 2003
Figure 1 Course of platelet counts, blast percentage and levels of WT1 transcripts during WT1 vaccination. The patient received repeated vaccinations with WT1 peptide and the adjuvants KLH and GM-CSF as indicated. The time course of the platelet counts (closed circles), percentage of blasts in bone marrow (columns) and quantitative levels of WT1 transcripts (WT1/PBGD, porphobilinogendeaminase; open circles) in bone marrow are shown. After the fourth vaccination and upon repeated analysis less than 5% of blasts were present paralleled by a 1–2 log reduction of WT1 transcripts and a normalization of platelet counts.
cycle of vaccination a drop of platelets to 52/nl was noted (Figure 1). Bone marrow analysis performed 4 weeks after the start of vaccination (prior to the third vaccination cycle) showed 30% blasts (Figure 1) and a leukemia relapse was diagnosed. Since the patient refused further chemotherapy, vaccination was continued. At 10 weeks after vaccination was initiated, the platelet count had increased to 65/nl (neutrophils 1.8/nl) and a bone marrow analysis showed less than 5% blasts. A complete hematological remission was confirmed 16 weeks after initiation of vaccination by bone marrow analysis when the platelets had increased to 135/nl, and has persisted for 12 þ months. Meanwhile, the patient has received a total 15 cycles of vaccination until February 2003. In this patient, without cytogenetic abnormality, we monitored minimal residual disease with real-time quantitative PCR analysis of WT1 transcripts in bone marow.7 WT1 levels in bone marrow and peripheral blood have been shown to be a sensitive marker to monitor myeloid leukemia.8 In our patient, levels of marrow WT1 transcripts paralleled the number of marrow blasts and were increased at the time of relapse (8.6 104), but returned to 10-fold lower levels (7.9 105) after four vaccinations (illustrated in Figure 1). Upon repeated analysis, WT1 levels have remained low and in the same range as levels detected in the bone marrow of healthy subjects.7 Potential side effects of vaccination with WT1 are of concern due to the low-level expression of WT1 in the kidney, and transiently during maturation in normal hematopoietic progenitor cells.9 There are, however, several preclinical and clinical studies showing WT1-specific immunity in the absence of autoimmunity (reviewed in Rosenfeld et al1 and Scheibenbogen et al10). In our patient, the complete blood count normalized upon remission and remained normal upon continuation of vaccination, indicating that no obvious impairment of normal CD34 þ stem cells had occurred. Retrospectively, the falling platelets during the first 6 weeks of vaccination were most likely Leukemia
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T-cell response analysis to WT1 126–134 peptide prior to and after vaccination
T-cell assay
Time of T-cell analysis (week and number of vaccinations received) Week 12
Tetramer Intracellular IFNg staining
a
0.08% 0.0%b
Week 8
Week 0
Week +4 (2)
Week +10 (4)
Week +16 (6)
Week +46 (15)
0.08% 0.0%
0.02% 0.15%
0.17% NDc
0.34% ND
0.54% 0.60%
0.92% 1.55%
a
Frequencies of CD3+CD8+ T cells binding to HLA-A0201-WT1 peptide 126–134 tetrameric complexes. Frequencies of CD3+CD8+ T cells specifically secreting IFNg in response toWT1 peptide 126–134. HIV peptide (reverse transcriptase protein, amino-acid position 476–484) was used as negative control, which was subtracted. c ND ¼ not done. b
due to the leukemic relapse and the patient had no evidence for an infection at this time. Serum creatinine and urea levels have been normal at all times during vaccination. A normal urine analysis was reported prior to the first and after the 15th vaccination. Side effects of GM-CSF (local erythema, induration and grade I/II fatigue) were observed. Monitoring of the T-cell response to WT1 126–134 was performed in peripheral blood using tetramer and intracellular IFNg staining by flow cytometry.10 Prior to vaccination, less than 0.1% of CD3 þ CD8 þ T cells stained with PE-labelled HLA-A0201WT1 peptide 126–134 tetrameric complexes (Beckman Coulter, San Diego) in peripheral blood samples obtained at three different time points (Table 1). After the fourth vaccination, a clearly enhanced fraction of 0.34% tetramer-binding T cells was detected, which further increased to 0.54% after the sixth and 0.92% after the 15th vaccination. In accordance with the results obtained with tetramers, p0.15% of CD3 þ CD8 þ T cells specifically produced IFNg in response to WT1 peptide 126–134 prior to vaccination, while 0.60 and 1.55% of CD3 þ CD8 þ T cells were detected after the sixth and 15th vaccination, respectively. In accordance with our observations, induction of complete remission by vaccination with proteinase 3 peptide has been reported recently in two patients with AML (Molldrem et al. American Society of Hematology Abstract Book 2002; 100, abstract 8). Both early observations from peptide vaccination trials in AML patients indicate an unexpected therapeutic efficacy and suggest that peptide vaccination may have a substantial therapeutic potential in hematological malignancies.
Acknowledgements This work is supported by a grant from the German Jose´-Carreras Foundation.
V Maila¨nder1 C Scheibenbogen1 E Thiel1 A Letsch1 IW Blau1 U Keilholz1
Leukemia
1
Charite´ – Universita¨tsmedizin Berlin, Campus Benjamin-Franklin, Medizinische Klinik III, Haematologie, Onkologie und Transfusionsmedizin, Berlin, Germany
References 1 Rosenfeld C, Cheever MA, Gaiger A. WT1 in acute leukemia, chronic myelogenous leukemia and myelodysplastic syndrome: therapeutic potential of WT1 targeted therapies. Leukemia 2003; 17: 1301–1312. 2 Menssen HD, Renkl HJ, Rodeck U, Maurer J, Notter M, Schwartz S et al. Presence of Wilms’ tumor gene (wt1) transcripts and the WT1 nuclear protein in the majority of human acute leukemias. Leukemia 1995; 9: 1060–1067. 3 Inoue K, Tamaki H, Ogawa H, Oka Y, Soma T, Tatekawa T et al. Wilms’ tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells. Blood 1998; 91: 2969–2976. 4 Oka Y, Elisseeva OA, Tsuboi A, Ogawa H, Tamaki H, Li H et al. Human cytotoxic T-lymphocyte responses specific for peptides of the wild-type Wilms’ tumor gene (WT1) product. Immunogenetics 2000; 51: 99–107. 5 Oka Y, Udaka K, Tsuboi A, Elisseeva OA, Ogawa H, Aozasa K et al. Cancer immunotherapy targeting Wilms’ tumor gene WT1 product. J Immunol 2000; 164: 1873–1880. 6 Scheibenbogen C, Schadendorf D, Bechrakis N, Nagorsen D, Hofmann U, Servetopoulou F et al. Effects of granulocyte– macrophage colony-stimulating factor and foreign helper protein as immunologic adjuvants on the T-cell response to vaccination with tyrosinase peptides. Int J Cancer 2003; 104: 188–194. 7 Siehl J, Thiel E, Heufelder K, Snarsk E, Schwartz S, Mailander V et al. Regulation of Wilms’ tumor gene 1(wt1) expression by the paired box genes PAX2 and PAX8 and by the hematopoietic transcription factor GATA-1 in human acute myeloid leukemias. Br J Hematol 2003; 123: 235–242. 8 Cilloni D, Gottardi E, De Micheli D, Serra A, Volpe G, Messa F et al. Quantitative assessment of WT1 expression by real time quantitative PCR may be a useful tool for monitoring minimal residual disease in acute leukemia patients. Leukemia 2002; 16: 2115–2121. 9 Menssen HD, Renkl HJ, Entezami M, Thiel E. Wilms’ tumor gene expression in human CD34+ hematopoietic progenitors during fetal development and early clonogenic growth. Blood 1997; 89: 3486–3487. 10 Scheibenbogen C, Letsch A, Thiel E, Schmittel A, Mailaender V, Baerwolf S et al. CD8 T-cell responses to Wilms tumor gene product WT1 and proteinase 3 in patients with acute myeloid leukemia. Blood 2002; 100: 2132–2137.
