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© 1999 American Society for Clinical Oncology Genetic Prodrug Activation Therapy for Breast Cancer: A Phase I Clinical Trial of erbB-2–Directed Suicide Gene ExpressionFrom the Imperial Cancer Research Fund Molecular Oncology Unit and Departments of Cancer Medicine and Histopathology, Imperial College School of Medicine, Hammersmith Campus, London, United Kingdom. Address reprint requests to Nicholas R. Lemoine, MD, Imperial Cancer Research Fund Molecular Oncology Unit, Imperial College School of Medicine, Hammersmith Campus, London W12 0NN, United Kingdom; email: n.lemoine{at}icrf.icnet.uk
PURPOSE: This trial was designed to test the safety and efficacy of a tumor-specific genetic prodrug activation therapy targeted by use of the human erbB-2 gene promoter. The erbB-2 oncogene is overexpressed in approximately 20% of cases of breast cancer and is associated with poor prognosis. PATIENTS AND METHODS: Twelve breast cancer patients received transcriptionally targeted gene therapy in a phase I clinical trial using direct intratumoral injection of plasmid construct combined with systemic administration of prodrug. The genetic prodrug activation therapy is specifically targeted to erbB-2–overexpressing breast cancer cells by use of a therapeutic cassette that contains the Escherichia coli cytosine deaminase gene driven by the tumor-specific erbB-2 promoter, thus allowing activation of fluorocytosine to the active cytotoxic fluorouracil only within tumor cells that express the oncogene. RESULTS: The approach was shown to be safe and to result in targeted gene expression in up to 90% of cases. Using a number of different assays, we demonstrated that significant levels of expression of the suicide gene were specifically restricted to erbB-2–positive tumor cells, confirming the selectivity of the approach. CONCLUSION: The results of this study, the first targeted gene therapy for breast cancer and the first to use the cytosine deaminase system in human subjects, are encouraging for the development of genetic prodrug activation therapies that exploit the transcriptional profile of cancer cells.
ALTHOUGH A SIGNIFICANT number of new anticancer agents have been developed and there has been some improvement in the overall survival rate for patients with breast cancer, the life expectancy for patients who develop metastases is limited. Therefore, there is an urgent need to develop novel systemic strategies for this major health problem. These strategies may be offered to patients to treat advanced disease that is resistant to chemotherapy and radiotherapy and may play an important role in improving adjuvant therapy for more limited disease. The challenge is to enhance the selectivity of systemic therapy so that tumor response is increased without undue toxicity to normal tissue. Genetic prodrug activation therapy (GPAT) uses transcriptional differences between normal and neoplastic cells to drive the selective expression of a metabolic "suicide gene" that is able to convert a nontoxic prodrug into its toxic metabolite. Genetically modified cells that express the nonmammalian enzyme cytosine deaminase (CD) gene are able to convert the nontoxic prodrug fluorocytosine (5-FC) to the toxic metabolite fluorouracil (5-FU), which inhibits RNA and DNA synthesis during the S phase of the cell cycle.1,2 Here we describe a transcriptionally targeted tumor-specific GPAT strategy using this system in patients with erbB-2–overexpressing breast cancer. Overexpression of erbB-2 is observed in approximately 20% of breast carcinomas and is associated with reduced relapse-free and overall patient survival.3 High erbB-2 receptor levels have been shown to correlate with poor prognosis in patients with nodal metastases of their breast cancer, and there is a trend toward shorter survival in node-negative disease.4-8 erbB-2 status has also been found to be predictive of resistance to endocrine and cytotoxic therapies.9-11 Overexpression of erbB-2 in breast cancer is a result of both increased gene transcription and gene amplification. The activity of the erbB-2 promoter is enhanced in overexpressing cells through binding of members of the AP-2 family of transcription factors to a response element within the proximal promoter. We have shown previously that a 500–base pair (bp) fragment of the proximal promoter (containing the AP-2 binding site) driving the CD gene resulted in levels of CD expression and cell death (upon exposure to 5-FC) directly proportional to the erbB-2 status of the cells when transduced into a panel of breast and pancreatic tumor cell lines using a retroviral vector.12 We now report an evaluation of intratumoral injection of a plasmid (pERCY) containing the same proximal erbB-2 promoter fragment/CD chimera and systemic infusion of the prodrug 5-FC. We conducted a study of 12 patients to assess the safety of the approach, the efficiency of in vivo gene transfer, and the functional expression of the suicide enzyme, and to find direct evidence of local antitumor activity in human tumor nodules caused by activation of 5-FC to 5-FU. The results show that the approach is safe, and expression of the therapeutic construct is observed in the majority of injected nodules. Expression at the mRNA and protein levels is detected in 10% to 85% of erbB-2–positive tumor cells, whereas no expression is evident in normal local epithelial and stromal tissues.
Clinical Protocol Twelve postmenopausal women with multiple well-demarcated, nonulcerating skin metastases less than 2 cm in diameter were recruited from a number of clinical centers in the United Kingdom. Before receiving any form of experimental therapy, medical history and tumor biopsy histology were reviewed, and the patients' clinical status was reassessed. Baseline studies were performed for hemoglobin, hematocrit, WBC count, platelet count, erythrocyte sedimentation rate, protein electrophoresis, renal function, liver enzymes, C-reactive protein, rheumatoid factor, antinuclear and anti–double-stranded DNA antibodies, immunoglobulin levels, and urinalysis. These tests were repeated 1 week after injection of plasmids. All patients had histologically proven cutaneous relapse of their breast cancer, good performance status (World Health Organization status 0, 1, or 2), normal renal and hematologic parameters, and a life expectancy of at least 3 months. Other prerequisites included failure of conventional treatment (radiotherapy, endocrine therapy, and at least one systemic form of chemotherapy) with an interval of at least 4 weeks since previous chemotherapy. Patients with visceral metastases, active autoimmune disease, or concomitant infection with hepatitis virus or human immunodeficiency virus were excluded. Patients continued to be reviewed after GPAT both in the Gene Therapy Clinic and by their own oncologist. The erbB-2 status was established by immunohistochemistry using two antibodies to the human erbB-2 receptor (Dako, High Wycombe, United Kingdom, and Biogen, Poole, United Kingdom), which gave concordant results. The positive control was tissue from a case of Paget's disease of the nipple, which is known to overexpress the erbB-2 oncogene. Four-micrometer sections were mounted onto poly-L-lysine–coated slides. Endogenous peroxidase activity was blocked by incubation in 0.3% H202 for 15 minutes, and the potential for nonspecific binding of erbB-2 antibodies was reduced by blocking in normal goat serum at 1/20 dilution for 30 minutes. Slides were incubated in primary antibodies that were diluted according to the manufacturer's recommendation at 4°C overnight, then incubated for 30 minutes at room temperature with the appropriate biotinylated secondary antibody before labeling with peroxidase-streptavidin at 1/500 dilution also for 30 minutes and subsequent incubation in diaminobenzidine chromogen for 10 minutes. Membrane immunoreactivity in at least 30% of the tumor cells was considered a positive result to qualify for trial entry. Only membrane-associated staining was considered significant because previous studies have shown that such immunoreactivity correlates with at least threefold overexpression of erbB-2 compared with that in normal breast.13 The degree of overexpression of erbB-2 by immunohistochemistry was designated according to the proportion of tumor cells that expressed the oncoprotein: moderate (+) indicated that up to 30% of all tumor cells were positive, intermediate (++) indicated that 30% to 70% were positive, and high (+++) indicated that more than 70% were positive.
For each patient, three similar-sized discrete lesions were marked, measured, and photographed: one lesion was injected with pERCY plasmid that contained the proximal erbB-2/CD chimera, and the second was injected with the control plasmid polyNeo, which had the same plasmid backbone as pERCY with the erbB-2 promoter/CD chimera removed (see Patients and Methods; Fig 1). The third lesion was not injected and served as another control. The clinical schedule is shown in Fig 2, and details of the plasmid dose escalation in the first four patients and prodrug infusion (in the first eight patients only) are listed in Table 1. Responses to GPAT were evaluated clinically by an independent observer (eg, changes in shape of the nodules), as well by two perpendicular measurements of the tumor nodule diameters and by clinical photography. Complete tumor response was defined as the complete disappearance of the injected cutaneous tumor nodule; partial response was defined as
All injections were in 0.2 mL of sterile water and directed into the center of the tumor nodules using a 22-gauge needle. A biopsy specimen was taken from the injected nodules at 24 hours, and an intravenous infusion of 5-FC prodrug 200 mg/kg/24 hours (Alcobon; ICN Pharmaceuticals, Basingstoke, United Kingdom) commenced 48 hours after the DNA injection for the first eight patients only, given for a total duration of 48 hours. Further biopsy specimens, measurements, and photographs were taken 7 days after the initial injection.
Clinical Safety
Construction of pERCY and polyNeo
Digestion of Therapeutic and Control Plasmids
Expression of CD
Detection of Intralesional CD mRNA by In Situ Hybridization
Thin-Layer Chromatography
A total of 72 patients with nodular cutaneous recurrence of breast cancer were screened for erbB-2 status; biopsy specimens from 14 (19%) of the screened patients met the criteria for overexpression of erbB-2. Of these 14 patients, 12 were able to participate in the study. Eleven patients had moderately or poorly differentiated invasive ductal carcinoma of the breast, and one patient had metastatic Paget's disease of the breast. The size of nodules varied between patients, but in each case the treated and control nodules were matched for size and shape. Histopathologic examination showed that the degree of stromal reaction was variable between samples. Clinical details of individual patients along with plasmid and prodrug doses are listed in Table 1. Within the group of 12 recruited patients, there was a wide range in terms of age (range, 34 to 78 years) and duration of disease (range, 6 to 36 months). Although five of the 12 patients had been premenopausal at the time of diagnosis, all patients were postmenopausal at the time of the trial. There were also differences in the extent of overexpression of erbB-2 as determined by immunohistochemistry. Each patient had received appropriate surgery, chemotherapy, radiotherapy, and hormonal therapy in their own clinical units. Five patients were receiving hormonal therapy with tamoxifen or a peripheral aromatase inhibitor that was continued during the gene therapy trial. The effects of GPAT on injected tumors are listed in Table 1. There was evidence of reduction of tumor volume after pERCY injection in four patients: one patient had received 200 µg of plasmid (patient no. 3), and the other three had received 400 µg of plasmid (patients no. 5, 9, and 12). In patient no. 3, the reduction in tumor volume was evident macroscopically with involution of the apex of the lesion without ulceration of the overlying skin, whereas the corresponding control lesions remained unchanged (Fig 3). In patient no. 5, there was also regression in the two control lesions (the uninjected nodule and the lesion injected with polyNeo). A reduction in tumor volume after pERCY injection was also observed in two patients (patients no. 9 and 12) who did not receive prodrug. Patient no. 4 showed some flattening of the pERCY-treated nodule without a change in overall volume of the lesion.
Analysis of Biopsy Tissue Immunohistochemistry for CD expression. Serial sections were used for histopathologic examination (hematoxylin-eosin staining) and immunohistochemistry with the anti-CD monoclonal antibody 16D8F2.15 The antibody was initially tested on the erbB-2–positive pancreatic cancer tumor cell line HPAF transfected with the CD gene (HPAF CD500) as well as the parental line (negative for CD; Figs 4A and 4B). For the biopsy samples, the degree of CD expression by immunohistochemistry was designated according to the proportion of immunoreactive tumor cells: moderate (+) indicated that up to 30% of all tumor cells were positive, intermediate (++) indicated that 30% to 70% were positive, and high (+++) indicated that more than 70% were positive. Control sections used phosphate-buffered saline in place of the primary antibody. The results are summarized in Table 2, and representative examples of the immunohistochemical studies are shown in Figs 4C through 4F. There was evidence of CD immunoreactivity in day-2 biopsy specimens from nine patients, and the enzyme was also detected, albeit at a lower level, 1 week later in the second biopsy specimen in three of these nine patients. In all cases, the CD immunoreactivity was restricted to tumor cells, with no detectable expression in stromal fibroblasts, endothelial cells, and mononuclear cells, or in the overlying epidermis. The localization of CD expression was consistent with erbB-2–expressing tumor cells (Fig 4I).
Detection of intralesional CD mRNA by in situ hybridization. In situ hybridization was performed on formalin-fixed tissue sections using 35S-labeled riboprobes transcribed from the cloned CD cDNA and beta-actin cDNA, and sections were exposed for emulsion for 11 days before the autographic sections were developed. A representative study is shown in Figs 4G and 4H. There was a positive signal in the first pERCY biopsy specimens from four patients. The degree of hybridization was designated according to the number of cells on each biopsy specimen that demonstrated a positive signal: moderate (+) indicated that up to 30% of all tumor cells were positive, intermediate (++) indicated that 30% to 70% were positive, and high (+++) indicated that more than 70% were positive. The results are listed in Table 2. No signal was observed in biopsy specimens from nodules from the same patients after injection with polyNeo. All four patients with positive signals on in situ hybridization also showed positive immunoreactivity for CD protein in their respective day-2 biopsy specimens. Determination of CD activity by thin-layer chromatography. This assay was validated initially on CD-transduced (HPAF CD500) and control (HPAF) pancreatic tumor cell lines before using biopsy tissue. This allowed us to determine the sensitivity of the technique by evaluating the number of cells required for detection of converted prodrug. One million cells from the CD-positive cell line HPAF CD500 were used in a pilot assay and were shown to give an easily detectable activity. In biopsy tissue from the clinical trial, there was evidence of enzyme activity by conversion of cytosine to uracil in three of 12 patients injected with the pERCY plasmid, and there was no conversion in any nodule injected with polyNeo. The degree of conversion varied among patients and is shown in Fig 5. In day-2 biopsy specimens, the conversion was 75% (patient no. 2), 69% (patient no. 6), and 8% (patient no. 8). Conversion was also observed in day-7 biopsy specimens of two patients, although to a lesser degree than that observed in the day-2 biopsy specimens (patient no. 2 at 44% and patient no. 5 at 8%). Despite the fact that the cryostat sections used for the CD immunohistochemistry were taken from the snap-frozen portion of the biopsy core before it was homogenized for the enzyme activity assay, there was no clear correlation with CD immunoreactivity on day-2 or day-7 biopsy specimens. Patient no. 6 showed evidence of 69% conversion of cytosine to uracil despite no evidence of CD immunoreactivity on either day-2 or day-7 biopsy specimens.
The present study demonstrates that a transcriptionally targeted GPAT strategy may be applied safely in the oncology clinic and without local or systemic complications. We have shown that local injection of plasmid DNA results in expression of the suicide gene and that the enzyme expressed in the tissue is capable of activating a prodrug. Targeted expression of a functional suicide gene in tumor tissue has been demonstrated by three different methods: in situ hybridization, immunohistochemistry, and thin-layer chromatography. There was evidence of gene transfer and expression in 11 of 12 patients, and, importantly, expression seemed to be highly selective for tumor cells. The high efficiency of gene transfer after simple intralesional injection of naked DNA in certain cases is particularly encouraging because no facilitators, such as liposomes, were used to increase plasmid uptake in the tumor nodules. We have shown for the first time that CD expression can be monitored in a human in vivo gene transfer study by immunocytochemistry using a CD-specific monoclonal antibody. Furthermore, thin-layer chromatography studies allowed us to evaluate the number of cells expressing functional CD that would then be capable of converting the prodrug to its toxic metabolite within the treated lesions. In two patients who received therapeutic gene and prodrug, there was evidence of tumor regression restricted to the nodule injected with the therapeutic construct (pERCY). In two other patients, there was evidence of a reduction in tumor nodule volume after pERCY injection (without prodrug infusion), which was also observed concurrently in a small proportion of other chest wall lesions, including those treated with polyNeo and uninjected lesions. A small degree of temporary regression in cutaneous disease is not an uncommon phenomenon in breast cancer and does not necessarily suggest an immune (ie, systemic) response induced by the plasmid DNA injection. However, several groups have reported that killing tumor cells in vivo with genetic prodrug activation systems (including CD) can lead to the development of T-cell–dependent antitumor immunity, probably by increasing the availability of tumor antigens to professional antigen presenting cells.2,18-22 The present study was performed in a relatively small number of patients, which partly reflects the restricted entry criteria for the trial. In addition, because the smaller scirrhous lesions were often the only treatable discrete tumors, it was difficult to obtain biopsy specimens later and sometimes little assessable tumor was available in the tissue. This complicated the analysis of gene transfer and expression in these cases. Theoretical risks of gene transfer include insertional mutagenesis, anti-DNA antibody formation, local infection, and tumor nodule ulceration. None of these complications occurred in any of these patients, despite the fact that relatively large doses of plasmid DNA was injected directly into the subcutaneous tissue, which reinforces our view that this is a safe strategy with minimal side effects. The completion of this trial highlights a number of practical issues that are clearly relevant for future trials that use GPAT or other gene therapy strategies. Selection of this specific phenotype of breast cancer (nodular cutaneous erbB-2–expressing lesions) caused some problems with patient recruitment. Despite the high prevalence of breast cancer and access to a large clinical practice at this unit, it took more than 18 months to recruit the 12 patients required for the trial. The advantages of using patients with skin nodules were ease of access, low risks associated with a skin biopsy, and the ability to monitor and assess lesions very simply. However, the densely fibrous nature of some of the lesions meant that the tissue that was injected with plasmid and/or underwent biopsy was not satisfactory for molecular analysis, and made histology, immunohistochemistry, and in situ hybridization difficult because of the low concentration of neoplastic cells in the matrix. Even under optimal conditions in vitro, a majority of cells can remain untransduced using currently available gene delivery technology. Alternatives include the use of viral vectors, but these may not be without their own complications; our experience suggests that this can lead to varying patterns of suicide gene expression that, in turn, will influence the efficacy of prodrug conversion. For example, studies have demonstrated that tissue-specific expression can often be lost as a result of promoter interference.23-25 As a consequence, improvements in promoter specificity and efficiency, along with improved design of insulated conditional expression elements in retroviral and adenoviral vectors, must be considered. Increasing local delivery of the therapeutic gene to tumor cells may be an approach appropriate to a single tumor in poorly accessible "sanctuary sites" such as in the brain, rather than the type of multiple separate metastases injected in this trial. However, even local delivery of high-dose suicide genes may not be sufficient for therapeutic effect, even by deliberate peritumoral injection. This has been exemplified by recent attempts at intratumoral delivery of a replication-defective retrovirus/suicide gene construct by intrathecal implantation of vector-producing cells to maintain high viral titers around the tumor. Very limited gene transfer occurred, despite high local density of virus-producing cells; only five of 15 patients with recurrent brain tumors in this study showed responses, and then only if tumors were very small.26 Further improvements to address these limitations include the use of viral vectors with the same proximal promoter element driving an alternative suicide gene such as HSV-TK, or the use of chimeric transcriptional elements that have the advantages of combining strong promoters with tissue-specific enhancers to improve targeting and suicide gene expression levels. We have shown that this approach has promise in a study that combined the erbB-2 and MUC1 promoter elements, resulting in increased sensitivity of MUC1-expressing cells to the effect of ganciclovir.27 Alternatively, concomitant expression of two suicide genes by one tissue-specific promoter could mediate greater levels of cytotoxicity beyond that observed with either suicide gene alone. There is recent evidence that, at least in vivo, the CD and HSV-TK suicide gene systems can be effectively combined not only to enhance cell kill, but also to increase the radiosensitivity of 9L gliosarcoma cells beyond that observed with each system alone.28 Future clinical trials may be able to exploit a bifunctional CD/HSV-TKsuicide gene coupled with radiotherapy to provide an improved means of treating cancer with a high degree of selectivity.
Supported by the Imperial Cancer Research Fund and the Mike Stone Cancer Research Fund. A.R. was supported by a clinical research fellowship from the Mike Stone Cancer Research Fund. We gratefully acknowledge the following clinical colleagues for contributing patients to the study: Drs Hilary Thomas, Claire Vernon, and Jonathan Waxman (Imperial College School of Medicine, Hammersmith Campus); Professor Charles Coombes (Imperial College School of Medicine, Charing Cross Campus); Dr Hani Gabra (Western General Hospital, Edinburgh); Dr Chris Gallagher (Royal London Hospital); and Dr Anne Hong (Royal Exeter Hospital). We also acknowledge the contribution made by Dr Jonathan Harris in the original construction of the pERCY and polyNeo plasmids and the preclinical work that validated the approach. In addition, we thank Professor Magnus von Knebel Doeberitz and Dr Karin Haak for the provision of the anti-CD antibody; Dr Craig Mullen for the CD cDNA; Karl-Heinz Blenk for technical assistance with the in situ hybridization; and Professor Bill Gullick and Dr Richard Vile for critical reading and discussion of the manuscript.
The contribution of each author is considered equal.
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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
50% reduction of the sum of the products of the perpendicular diameters. Biopsy specimens were collected after treatment using a 3-mm punch biopsy on days 2 and 7. The tissue was orientated and divided longitudinally using a sterile scalpel. One half of the tissue was snap-frozen in liquid nitrogen, and the other half was fixed in formalin and paraffin embedded the same day. 
