Abstract
Background: The object of this study was to investigate the safety and clinical response of immunotherapy targeting the WT1 (Wilms' tumor 1) gene product in patients with gynecological cancer. Patients and Methods: Twelve patients with WT1/human leukocyte antigen (HLA)-A*2402-positive gynecological cancer were included in a Phase II clinical trial of WT1 vaccine therapy. In all the patients, the tumors were resistant to standard therapy. The patients received intradermal injections of a HLA-A*2402-restricted, modified 9-mer WT1 peptide every week for 12 weeks. Tumor size, which was measured by computed tomography (CT), was determined every 4 weeks. The responses were analyzed according to Response Evaluation Criteria in Solid Tumors (RECIST). Results: The protocol was well tolerated; only local erythema occurred at the WT1 vaccine injection site. The clinical responses were as follows: stable disease (SD) in 3 patients and progressive disease (PD) in 9 patients. No patients had a complete (CR) or partial response (PR). The disease control rate was 25.0%. Conclusion: Although a small, uncontrolled, nonrandomized trial, this study showed that WT1 vaccine therapy for patients with gynecological cancer was safe and produced a clinical response.
Although there are well-established surgical, chemotherapeutic and radiotherapeutic treatments for gynecological cancer, the need for molecular-target therapy has increased, especially for recurrent disease that has acquired radio- or chemoresistance. With the rapid development of high-throughput techniques for identifying novel specific molecular targets in human cancer over the past few years, attention to targeted cancer therapy has dramatically increased. There has been a rapid increase in the identification of targets that have potential therapeutic application. The number of agents under preclinical and clinical investigation has grown accordingly. This emphasis on molecular biology has also resulted in significant changes in the treatment of gynecological malignancies.
Recent advances in tumor immunology have resulted in the identification of a large number of tumor-associated antigens that could be used for cancer immunotherapy, since their epitopes associated with human leukocyte antigen (HLA) class I molecules were recognized by cytotoxic T lymphocytes. One of the identified tumor associated antigens was the product of the Wilms' tumor gene, WT1 (1, 2).
WT1 was isolated as a gene responsible for a childhood renal neoplasm, Wilms' tumor (3, 4). This gene encodes a zinc finger transcription factor and plays an important roles in cell growth and differentiation (5, 6). Although the WT1 gene was categorized at first as a tumor-suppressor gene, it has recently been demonstrated that the wild-type WT1 gene performed an oncogenic rather than a tumor-suppressor function in many kinds of malignancies (7). The WT1 gene is highly expressed in various types of cancer, including gynecological cancer (8, 9).
We have performed a Phase I clinical trial to examine the safety of a WT1-based vaccine, as well as the clinical and immunological response of patients with a variety of cancer types, including leukemia, lung cancer and breast cancer (10). The WT1 peptide vaccine emulsified with Montanide ISA51 adjuvant and administered at a dosage of 0.3, 1.0, or 3.0 mg at 2-week intervals was safe for patients other than those with myelodysplastic syndromes. Furthermore, it has been confirmed that the potential toxicities of the weekly WT1 vaccination treatment schedule (3.0 mg per person) with the same adjuvant were also acceptable (11). To date, clinical response to WT1 peptide-based immunotherapy in Phase II trials with the weekly WT1 vaccinations has been reported for renal cell carcinoma (12), multiple myeloma (13) and glioblastoma multiforme (14).
In the present study, the clinical response to peptide-based immunotherapy targeting the WT1 gene product in patients with gynecological cancer was investigated.
Patients and Methods
The WT1 peptide. The immunization consisted of an HLA-A*2402-restricted, modified 9-mer WT1 peptide (amino acids 235-243 CYTWNQMNL), in which Y was substituted for M at amino acid position 2 (the anchor position) of the natural WT1 peptide. This variant induces stronger cytotoxic activity than the natural peptide (15). The WT1 peptide [Good Manufacturing Practice (GMP) grade] was purchased from Multiple Peptide Systems (San Diego, CA, USA) as lyophilized peptide.
Trial protocol. The entry criteria were as follows: 16-79 years of age; expression of WT1 in the cancer cells determined by immunohistochemical analysis; HLA-A*2402-positivity; estimated survival of more than 3 months; performance status 0-1; no severe organ function impairment and the written informed consent of the patient. At least 4 weeks prior to immunotherapy the patients were free from antitumor treatments such as surgery, chemotherapy and radiation. Patients with brain metastasis were excluded. The protocol was approved by the Institutional Review Board and the Ethical Committee at Kanazawa University.
Vaccination. The patients were first tested for an allergic reaction by injecting 30 μg of the peptide in saline intradermally. None of the patients exhibited immediate hypersensitivity. The injected site was also examined for delayed type hypersensitivity (DTH) response after 48 hours. Blood and urine tests to examine adverse events preceded every immunization. The patients received intradermal injections of 3.0 mg of HLA-A*2402-restricted modified 9-mer WT1 peptide emulsified with Montanide ISA51 adjuvant (SEPPIC S.A., Paris, France). The WT1 vaccinations were scheduled to be given weekly for 12 consecutive weeks.
Immunohistochemical analysis. Positive immunostaining of WT1 protein in the patient's tumor was a mandatory requirement for entry into the trial. A standardized staining protocol was adopted from a preceding trial (8). Briefly, formalin-fixed and paraffin-embedded tissue sections were first autoclaved in order to expose antigenic epitopes and then stained with polyclonal rabbit anti-WT1 IgG antibodies (C-19, sc-192; Santa Cruz Biotechnology, Santa Cruz, CA, USA) followed by a Vectastain abidin-biotin-peroxidase complex (ABC) kit (Vector Laboratories, Burlingame, CA, USA). Staining with a more specific monoclonal antibody, 6F-H2 (Dako, Glostrup, Denmark), was also performed and the results were consistent with those obtained with the polyclonal antibodies.
Evaluation of toxicity. Toxicities were evaluated according to the National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) ver. 3.0 (16). If an adverse event of grade 2 or 3 continued, further immunization was suspended until the problem was solved. An adverse event of grade 4 forced the immediate termination of the immunotherapy.
Evaluation of clinical response. After the WT1 vaccine was administered 12 times, the antitumor effect of the treatment was assessed by determining the response of the target lesions on computed tomography (CT) images. The tumor size was analyzed according to Response Evaluation Criteria in Solid Tumors (RECIST) (17), with results reported as complete response (CR), partial response (PR), stable disease (SD) or progressive disease (PD). The response rate was calculated as the percentage of the number of patients in which there was a CR or PR divided by the total number of patients. The disease control rate was calculated as the percentage of the number of patients in which there was a CR or PR or SD divided by the total number of patients.
Results
Patients characteristics. During the trial period, 28 patients were assessed for inclusion in the trial. Twenty-three out of the 28 patients (82.1%) had a WT1-positive tumor, as determined by immunohistochemical analysis. Because HLA-A*2402-restricted WT1 peptide was used, 11 patients with HLA-A*2402-negative type were excluded. Finally, 12 patients were enrolled in this study (Table I). The mean age of the 12 enrolled patients was 55.3 years (range 43-69 years). Out of the 12 patients, 9 had recurrent disease and 3 had disease progression after initial therapy. All the patients had received chemotherapy with or without radiotherapy.
Clinical response to vaccination. All the treated patients had a local inflammatory response with erythema at the WT1 vaccine injection site. No Grade 3 or 4 toxicities were observed.
A summary of the patient response to WT1 immunotherapy is shown in Table II. Clinical responses included SD in three patients and PD in nine patients, including three who dropped out of the trial due to tumor progression and poor general condition (Patients #1, #5 and #6). The patients who had an effective response continued to receive vaccinations until tumor progression was demonstrated.
Characteristics of all enrolled patients.
Clinical results in all enrolled patients.
The disease control rate in the initial 3 months (the clinical trial period) was 25.0%. One patient (Patient #12) experienced CR on a non-target lesion (Figure 1).
Discussion
The WT1 gene is physiologically expressed in some organs, such as the kidney, bone marrow and pleura. Experimental evidence shows that WT1-specific cytotoxic T lymphocytes kill WT1-expressing tumor cells without killing normal cells (18). Consistent with these data, in the present study, all the treated patients had an inflammatory response with erythema at the WT1 vaccine injection site, but no systemic toxicities were observed. Taken together, these findings allow the conclusion that the WT1 vaccination was safe, and the patients tolerated it well.
The internationally approved RECIST guideline, was originally developed for the evaluation of chemotherapy (17). However, peptide immunotherapy, especially if peptide is administered alone without adjuvant, may not lead to such a drastic tumor regression as chemotherapy. It is probable that some cancer patients treated with cancer vaccines can survive long-term without remarkable tumor regression (14). Their tumors could be stabilized or could regress following a temporary increase in size after vaccination since, in general, peptide-based immunotherapy does not act as quickly as chemotherapy due to the time needed to induce lymphoid activation. In the present study, a decrease in tumor size and normalization of the level of tumor marker were observed about 2 months after the initial WT1 vaccination (Patient #12 Figure 1). For this reason, it might be allowable to modify the RECIST guideline according to peptide-based immunotherapy. If for instance, the baseline of the sum of the longest diameters of the target lesions was shifted to 1 month after the initial WT1 vaccination, the disease control rate became as high as 41.7% (Table III).
Clinical results in all enrolled patients when baseline of SLD* was changed.
Patient 12: Arrows in computed tomography indicate primary tumor mass. SLD, Sum of the longest diameters; RV, reference value.
On the other hand, peptide immunotherapy has several advantages over chemotherapy. Side-effects are almost absent except for skin reaction. The quality of life of patients is generally very good. Compared with stronger side-effects and the eventual development of resistant tumors as is often observed in chemotherapy, this seems to be a clear benefit. Therefore, when vaccination-induced clinical responses are evaluated with RECIST, which is a gold standard in the field of cancer chemotherapy, it may be recommended that SD is highly regarded in cancer immunotherapy, particularly when SD persists long-term. In order to evaluate these advantages of peptide-based immunotherapy, it may be better to develop appropriate criteria for evaluation of immunotherapy (19).
Although there were no patients with CR or PR, the disease control rate (patients with SD) of 25.0% was favorable. Identification of effective new agents is difficult in patients who have previously been treated with standard therapy because response rates to agents, even of known efficacy, are known to be lower than in previously untreated patients. The WT1 vaccination, however, had disease-stabilizing, as well as disease progression-inhibiting, effects with systemic toxicity that was much less than that of chemotherapy or radiotherapy, and thus allowed the vaccinations to be given for a long time.
In patients with advanced cancer, the basal metabolic rate declines and cachexia occurs. Cachexia is often associated with breakdowns in the host immune system. Moreover, cluster of differentiation (CD)4+CD25+ forkhead box P (FOXP)3+ regulatory T-cells are elevated in malignancy and can thwart protective antitumor immunity (20). In this study, the activity of WT1 peptide alone was examined and adjuvant that would activate dendritic cells and/or helper T-cells was not included. The use of a more suitable adjuvant, such as bacillus Calmette-Guerin cell-wall skeleton (BCG-CWS) (21), granulocyte-macrophage colony-stimulating factor (GM-CSF) (22, 23), CpG (24), interferon-α (25) and interleukin-2 (26) may further enhance the clinical usefulness of this treatment for patients with gynecological cancer.
Although a small, uncontrolled, nonrandomized trial, this study showed that WT1 vaccine therapy for patients with gynecological cancer was safe and produced a clinical response. Based on these results, further clinical studies of WT1 vaccine therapy in patients with gynecological cancer are warranted.
Acknowledgements
This work was supported by a Grant-in-Aid for Young Scientists (B) and (A) (No. 19791140 and No. 21689044, respectively) from the Ministry of Education, Culture, Sports, Science and Technology, of the Japanese Government. We appreciate the following members for their cooperation in this clinical trial: Drs. M. Tanaka, M. Takakura, Y. Maida, M. Hashimoto, N. Mori, Y. Mizumoto, T. Ikoma and R. Yamazaki (Kanazawa University Graduate School of Medical Science); Drs. T. Tsuchida and M. Kato (Fukui Prefectural Hospital); Dr. T. Kohama (Keiju Medical Center); Drs. R. Yamada and S. Hirabuki (Ishikawa Prefectural Central Hospital); Dr. T. Kanaya (National Hospital Organization Kanazawa Medical Center); Dr. S. Waseda (Kanazawa Medical University); Dr. R. Kawahara (Koseiren Takaoka Hospital) and Dr. Y. Yamakawa (Saiseikai Takaoka Hospital). In addition, we would like to thank Mses. T. Umeda, H. Nakajima, T. Hakamata and C. Yoshikawa for their technical assistance and coordination of the clinical research.
- Received June 24, 2009.
- Revision received October 8, 2009.
- Accepted October 13, 2009.
- Copyright© 2009 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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