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Keywords:

  • gene therapy;
  • granulocyte-macrophage colony-stimulating factor;
  • renal cell carcinoma;
  • vaccine

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Case report
  5. Discussion
  6. References

A phase I study of granulocyte-macrophage colony-stimulating factor (GM-CSF) gene-transduced tumor vaccine for patients with metastatic renal cell carcinoma (RCC) was initiated in 1998, as the first cancer gene therapy in Japan. The study is still ongoing, but the first patient is presented here as a case report. The patient was a 60-year-old man with Stage IV CRC with multiple lung metastases. After surgical resection of the tumor, autologous tumor cells were transduced and cultured to produce GM-CSF. The patient received a total of 2.2 × 108 gene-transduced autologous vaccine cells by subcutaneous injection. During the course of vaccination, growth of the largest metastatic mass slowed to some extent; however, multiple new lesions developed. About 1 month after the start of low-dose IL-2 therapy, rapid and remarkable regression in a large lung hilar metastatic mass was noticed. The patient died of progressive disease 7 months after the start of GM-CSFgene therapy. Careful histological examination by autopsy revealed that the responding mass was infiltrated by CD8 positive dominant T lymphocytes, and did not exhibit vasculitis or any other changes associated with active autoimmune disease.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Case report
  5. Discussion
  6. References

Treatment options for metastatic renal cell carcinoma (RCC) are limited due to the lack of effective systemic therapy. According to preclinical animal studies, vaccination using autologous granulocyte-macrophage colony-stimulating factor (GM-CSF) transduced tumor cells is considered to be one of the challenging immunotherapies for RCC.1 In April 1998, we submitted our preclinical data on cell processing and gene transfer to the Institute of Medical Science, University of Tokyo (IMSUT) Institutional Review Board. In August 1998, the Japanese government granted permission to initiate gene therapy. Within the same year,the phase I study of a GM-CSF gene-transduced tumor vaccine for patients with metastatic RCC began as the first cancer gene therapy in Japan.2 The study is currently being conducted as a multi-institutional study organized by IMSUT. The present report describes the clinical course of the first patient who received the GM-CSF gene therapy.

Case report

  1. Top of page
  2. Abstract
  3. Introduction
  4. Case report
  5. Discussion
  6. References

Clinical course

A 60-year-old man with gross hematuria was seen at Tsukuba University Hospital (TUH) in September 1998. The patient was diagnosed as stage IV RCC (UICC classification 1997) with multiple lung metastases (Fig. 1). Following careful screening for eligibility criteria and after providing written informed consent, the patient was transferred to the Research Hospital of IMSUT, where he was enrolled in a phase I study of GM-CSF gene therapy. A 10 cm RCC tumor arising from the upper pole of the right kidney was resected, and the cell processing began on the same day. The largest part of resected tumor consisted of clear cell carcinoma with nuclear grade 2 as shown in Fig. 2a. The autologous RCC cell culture from 80 g of tumor tissue was successfully propagated, and the human GM-CSF gene was introduced using the MFG-S retrovirus vector.1–4 Four-hundred and sixty million transduced cells with GM-CSF production of 49 ng/106 cells per 24 h were harvested. The harvested vaccine cells were lethally irradiated with 150 Gy of X-ray, then frozen in aliquots and stored in liquid nitrogen. BioReliance (Rockville, Maryland,USA) conducted standard safety testing according to US Food and Drug Administration guidelines and confirmed that culture supernatants were free of replication-competent retrovirus (RCR).

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Figure 1. Plain chest X-ray showing a large metastatic mass in the right hilar region and multiple metastases in both lungs.

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Figure 2. H&E staining × 100 of the (a) nephrectomised primary lesion, (b) lung metastatic lesions and (c) right lung hilar metastasis.(d) Immunohistochemical staining with anti-CD8 antibody of the right hilar mass (× 100). The largest part of the primary site consisted of clear cell carcinoma (a), whereas most metastatic sites consisted of granular cell carcinoma (b). The right lung hilar metastasis (the nodule that responded after IL-2 therapy) showed diffuse necrotic change (c). Immunohistochemistry revealed CD8 positiveT-lymphocyte dominant infiltration of the right lung hilar metastasis(d).

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After safety and release testing, GM-CSF gene therapy was initiated by subcutaneous injection of vaccine cells. From December 1998 toApril 1999, the patient was injected with a total of 2.2 × 108 vaccine cells divided into ten doses (4 × 107 cells in the first injection, followed by 2 × 107 cells at 2-week intervals). The patient experienced no remarkable side-effects except for low-grade fever, and local erythema and in duration at the vaccination sites, both of which resolved spon­taneously. Laboratory tests revealed no remarkable changes except transient eosinophilia up to 700 cells/mm3, which peaked 48 h after vaccination and returned to normal the next day. There was no significant alteration in the serum GM-CSF level after vaccination. During the course of vaccination, the delayed-type hypersensitivity (DTH) reaction was examined by the intradermal injection of 1 × 106 lethally irradiated cultured autologous RCC cells (non-transduced RCC cells) as an in-vivo immunological examination.3 As a control, the same number of lethally irradiated cultured autologous normal kidney cells, which were prepared from normal part of nephrectomy tissue, were injected intradermally. Reactions to DTH increased markedly after the start of vaccination; however, weak reaction was also observed at the site of normal kidney cell injection.

After therapy began, the growth of the largest lung hilar mass,as evaluated by helical computed tomography (CT), had slowed to some extent; however, new multiple nodules appeared in other lung fields. The patient maintained good performance status until the 10th injection, but routine radiographic examination at that time revealed development of new metastases in the liver and brain. At this point, vaccination was discontinued, and the patient was transferred to TUH for further treatment and supportive care. No RCR was detected in blood samples during or after the end of vaccination.

After transfer to TUH, a symptomatic brain metastases were treated with a single session of gamma-knife radiosurgery for prevention of central nervous system complications. Intravenous administration of human recombinant interleukin (IL)-2 (Teceleukin, Shionogi, Osaka), at a daily dose of 0.7–1.4 × 106 Japan Reference Unit (JRU), began 1 month after the end of the vaccination period. A few weeks after the start of IL-2 therapy, chest X-ray revealed a decrease in the size of the right hilar large mass, despite new metastatic deposits and pleural effusion in both lungs. The findings were confirmed by chest CT scan 1 month after the start of IL-2 therapy (Fig. 3). Treatment continued for 5 weeks for a total IL-2 dose of 20 × 106 JRU. However, the patient's condition deteriorated due to progression of metastases. The patient died of multiple RCC metastases 9 months after nephrectomy and 7 months after the start of GM-CSF gene therapy.

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Figure 3. Chest CT scan (a) before and (b) 1 month after the initiation of IL-2 treatment. Note the marked regression of a right lung hilar mass despite progression of the other metastatic nodules and pleural effusion.

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Autopsy and histological findings

With permission from the family, an autopsy was performed 5 h after the patient's death. Grossly massive RCC metastases were found in both lungs and the liver. Widespread metastases were also found in the pancreas, duodenum, left kidney, pleura and retroperitoneal lymph nodes. Histological examination of the metastasis sites showed that almost all examined nodules consisted of granular cell carcinoma, which was in sharp contrast to the histology of the primary site where clear cell carcinoma was dominant (Figs 2a,b). In addition, sarcomatoid changes were observed in the kidney metastasis. These findings indicate the apparent histological divergence of metastatic sites. While scattered necrotic foci were seen, RCC cells were morphologically viable in most of the metastatic sites. In contrast, histology of the right lung hilar metastasis, the lesion that shrank after IL-2 therapy, showed diffuse necrotic changes (Fig. 2c). Viable cells were barely seen in pseudocapsule of this lesion as bizarre foci of granular cell carcinoma. No significant differences were found in the degree of lymphocyte infiltration and dendritic cell infiltration; however, the shrunk lesion was infiltrated with proportionately more CD8 positive T lymphocytes, compared to other non-responding metastatic lesions, as shown in Fig. 2d. The similar CD8 positive T-lymphocyte accumulation was seen in one necrotic deposit in the liver. Careful histological examination did not reveal vasculitis or any other changes associated with active autoimmune disease.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Case report
  5. Discussion
  6. References

Since RCC is thought to be an immunogenic cancer, gene immunotherapy is considered to be one of the challenging gene therapy strategies. In 1997, Simons et al. reported a phase I study of autologous RCC vaccination in the USA.3 An objective partial response was observed in one of seven patients treated with GM-CSF gene-transduced tumor vaccine.3 In Japan, a phase I study of a GM-CSF gene-transduced tumor vaccine for metastatic RCC patients (the first cancer gene therapy in the country) is currently being conducted as a multi-institutional study organized by IMSUT. The procedure for producing autologous vaccine cells is conducted at IMSUT under Good Manufacturing Practices. Although the treatment protocol is essentially the same as the one reported by Simons et al.,3 the vaccination schedule is longer in the current study than in the previous clinical trial conducted in the USA.

The objectives of the current study are to test the safety of the gene therapy and evaluate immunological anti-tumor effects. In the present paper we reported the clinical course of the first patient enrolled in the study. During and after gene therapy, no remarkable side-effects were observed by physical examination or laboratory tests. In addition, no pathological findings specific to gene immunotherapy, such as autoimmune disease,were found by autopsy. To our knowledge, this is the first detailed report of an autopsy of a patient treated with GM-CSF gene therapy.

Despite the strong positive DTH conversion against autologous RCC cells after vaccination, the clinical response of the GM-CSF gene therapy resulted in progressive disease. However, the subsequent low-dose IL-2 therapy showed rapid and remarkable regression in a large lung hilar metastatic mass. The most striking histological finding in the responding mass was CD8 positive T-cell infiltration. It suggests the CD8 positive T-cell might be an important effect or cell in regression of the mass. Since the mass regressed after IL-2 therapy, it is not certain that the GM-CSF gene therapy plays a role in this immune response. Further clinical and immunological evaluation of the other patients enrolled in the study is required.

Finally, there was a difference in RCC histology between the nephrectomised primary site and the metastatic sites. This may have been the result of natural selection of metastatic phenotype, because it is well known that metastases from clear cell at the primary site are not always clear cell and may be considerably less differentiated than cells at the primary site. Another possibility is the selective escape of non-clear cell populations from immunity induced by vaccine prepared from clear cell carcinoma, which was dominant at the primary site. Although it is not clear whether selective escape might be involved in the present case, the genetic instability and heterogeneous features of RCC may require additional treatment combined with immune gene therapy. At the time of this report, the trial is in progress, and an additional three patients have been successfully treated with no significant side-effects. Further clinical and immunological evaluation including these patients is required to ascertain the clinical benefits of GM-CSF gene therapy in metastatic RCC.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Case report
  5. Discussion
  6. References
  • 1
    Dranoff G, Jaffee E, Lazenry A et al. Vaccination with irradiated tumor cells engineered to secrete murine granulocyte-macrophage colony-stimulating factor stimulates potent, specific, and long-lasting anti-tumor immunity. Proc. Natl Acad. Sci. USA 1993; 90: 353943.
  • 2
    Tani K, Nakazaki Y, Hase H et al. Progress reports on immune gene therapy for stage IV renal cell cancer using lethally irradiated granulocyte-macrophage colony-stimulating factor-transduced autologous renal cancer cells. Cancer Chemother. Pharmacol. 2000; 46 (Suppl.): S736.
  • 3
    Simons JW, Jaffee EM, Weber CE et al. Bioactivity of autologous irradiated renal cell carcinoma vaccines generated by ex vivo granulocyte-macrophage colony-stimulating factor gene transfer. Cancer Res. 1997; 57: 153746.
  • 4
    Jaffee EM, Dranoff G, Cohen LK et al. High efficiency gene transfer into primary human tumor explants without cell selection. Cancer Res. 1993; 53 (Suppl.10): 22216.