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-A0201- and 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, Immucothel®, biosyn, Germany) as adjuvant injected i.d. and s.c. on day 0. GM-CSF (Leukomax®, Essex Pharma, Germany) in a dose of 75 μg per day was injected s.c. at the same site (proximal thigh) as the WT1 peptide on days –2 to +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 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+ 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 × 10−4), but returned to 10-fold lower levels (7.9 × 10−5) 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+ stem cells had occurred. Retrospectively, the falling platelets during the first 6 weeks of vaccination were most likely 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 IFNγ staining by flow cytometry.10 Prior to vaccination, less than 0.1% of CD3+CD8+ T cells stained with PE-labelled HLA-A0201-WT1 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, 0.15% of CD3+CD8+ T cells specifically produced IFNγ in response to WT1 peptide 126–134 prior to vaccination, while 0.60 and 1.55% of CD3+CD8+ 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.
This work is supported by a grant from the German José-Carreras Foundation.
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Strategies to Reduce Relapse after Allogeneic Hematopoietic Cell Transplantation in Acute Myeloid Leukemia
Current Hematologic Malignancy Reports (2013)