Gene therapy for carcinoma of the breast: Therapeutic genetic correction strategies
© Current Science Ltd 2000
Received: 2 November 1999
Accepted: 24 November 1999
Published: 17 December 1999
Gene therapy is a therapeutic approach that is designed to correct specific molecular defects that contribute to the cause or progression of cancer. Genes that are mutated or deleted in cancers include the cancer susceptibility genes p53 and BRCA1. Because mutational inactivation of gene function is specific to tumor cells in these settings, cancer gene correction strategies may provide an opportunity for selective targeting without significant toxicity for normal nontumor cells. Both p53 and BRCA1 appear to inhibit cancer cells that lack mutations in these genes, suggesting that the so-called gene correction strategies may have broader potential than initially believed. Increasing knowledge of cancer genetics has identified these and other genes as potential targets for gene replacement therapy. Initial patient trials of p53 and BRCA1 gene therapy have provided some indications of potential efficacy, but have also identified areas of basic and clinical research that are needed before these approaches may be widely used in patient care.
Keywordsadenovirus BRCA1 cancer gene therapy p53 retrovirus
Because cancer is a somatic genetic disease, it should not be surprising that many gene therapy protocols have been designed to treat various forms of cancer. Even hereditary cancer syndromes generally include a required somatic gene alteration. For example, patients with hereditary breast and ovarian cancer due to BRCA1 mutations are born with a mutation in one BRCA1 allele, but only develop cancer after mutation or allelic loss of the other BRCA1 allele. Thus, the complete genetic disorder is only present in the tumor cells that are targeted for somatic gene therapy. This is in contrast to inherited germline diseases, in which the genetic defect is present in all cells.
One strategy for cancer gene therapy is that of genetic correction. Because known gene defects are present exclusively in the cancer cells, genetic correction therapies provide a potential rational therapy that is molecularly targeted to cancer cells, but should not necessarily affect normal nonmalignant cells. A number of genetic correction strategies have been designed to treat cancer, including correction of p53 and BRCA1 [1*,2,3]. This review discusses some of the scientific information related to genetic correction strategies and then describes animal models and patient trials designed to initially test this therapeutic approach.
p53 is the most commonly mutated tumor suppressor gene in solid tumors and is also mutated in the germline of patients with the rare hereditary Li-Fraumeni syndrome . The p53 gene is specifically relevant to the development and progression of breast cancer, because p53 is frequently mutated in breast cancer specimens and Li-Fraumeni syndrome patients develop breast cancer as part of their multiple cancer syndrome . Thus, p53 genetic correction is a rational approach for breast cancer, particularly in those rare patients with breast cancer as part of Li-Fraumeni syndrome. The function of wild-type p53 is suppression of cell proliferation through a multiprotein regulatory pathway that is focused around the retinoblastoma gene and control of apoptosis . Because p53 may naturally function as an inhibitor of cell proliferation, it inhibits cell growth in most normal and malignant cells [6,7], with few exceptions . For this reason it may effectively inhibit tumor growth even in cancers that do not have p53 mutations [6,7]. Preclinical animal studies of adenovirus-based p53 gene therapy for cancer in both cell culture and animal models [9,10,11] have demonstrated tumor suppression. Clinical trials of p53 gene therapy for lung cancer[1*,12*] have been reported, and these studies demonstrate gene transfer of adenoviral-p53, induction of apoptosis, and some indication of therapeutic response. In a phase 1 clinical trial of nonsmall cell lung cancer [12*], 8% of treated patients showed a partial response and 64% of patients showed disease stabilization ranging in duration from 2 to 14 months.
Although the molecular function of BRCA1 is controversial and may include DNA repair or transcriptional functions , overexpression of BRCA1 into sporadic breast or ovarian cancer cells, which usually show low BRCA1 expression , results in growth inhibition and tumor suppression [15,16,17,18,19,20,21,22,23]. Growth inhibition by BRCA1 has itself been controversial, leading some to question the rationale for BRCA1 gene therapy , but subsequent studies have shown growth inhibition or tumor suppression by BRCA1 in vitro [16,17,19,20,21,22,23] and in vivo [15,17,21,25]. The mechanism of growth inhibition by BRCA1 is unknown and may involve interactions with WAF1/CIP1 , p53 , Rb  or induction of apoptosis [18,19].
Although BRCA1 is only mutated in a small percentage of breast or ovarian cancers, the majority of sporadic breast and ovarian cancers appear to express low levels of BRCA1 messenger RNA and protein [14,26,27,28,29,30]. This appears to be a consequence of loss of heterozygosity and promoter methylation of the remaining BRCA1 allele [14,26,27,28,29,30,31,32,33,34,35]. This finding is important because it indicates that restoration of normal 'wild-type' BRCA1 expression levels in many sporadic cancers may inhibit tumors by a 'genetic correction' strategy, wherein the loss of BRCA1 expression contributes to tumorigenesis. These results constitute the scientific basis for testing BRCA1 gene therapy in patients with sporadic breast, ovarian, and prostate cancers that lack specific point mutations in the BRCA1 gene.
Several different BRCA1 viral vectors have been constructed and tested for efficacy in preclinical xenograft models of breast and ovarian cancer. Initial studies of a BRCA1 retroviral vector employed a complementary DNA that encoded a splice variant vector that eliminates the first 71 amino acids of the human protein, termed BRCA1sv [2,25,36*]. Studies of both growth inhibition and DNA repair do not identify cellular or molecular differences between BRCA1sv and BRCA1 complementary DNAs [36*,37,38,39,40]. Intaperitoneal injection of either BRCA1sv or a full-length BRCA1 retroviral vector into ovarian cancer or breast cancer xenografts in nude mice produces tumor inhibition [2,15,25,36*,37,38,39]. These studies show that treatment of established SKOV3 or PA-1 ovarian cancer nude mice xenografts with either the full-length or the splice variant BRCA1 retroviral vector results in tumor suppression. Necropsies showed that PA-1 tumor-bearing mice treated with control media or low-dose LXSN-BRCA1sv died with large intra-abdominal tumors and ascites, whereas mice with high-dose LXSN-BRCA1sv treatments died of lung metastasis with significantly smaller abdominal tumor. The PA-1 tumor model is an established xenograft model for ovarian cancer [37,38,39,41,42].
The published phase 1 trial of BRCA1sv retroviral gene therapy  demonstrated gene transfer and expression of the intraperitoneally injected LXSN-BRCA1sv vector. The vector was moderately stable in the peritoneum of these patients, and antibody formation was rare. The phase 2 trial performed in a group of patients with lower tumor burdens [36*], however, demonstrated that tumor size and immune status strongly influence patient response to retroviral vectors, and that vectors packaged in mouse cells are not sufficiently stable to treat patients with small volume intraperitoneal ovarian cancer.
Patient model systems for breast and ovarian cancer
The initial patient trials of p53 and BRCA1 gene therapy demonstrated the potential for gene therapy, but also clearly demonstrated both basic and clinical research that is needed before these therapies can be successful in patients. Initial human clinical trials of BRCA1 retroviral gene therapy taught us that the approach was safe, but that healthy patients developed immune responses towards retroviral vector therapy that decreased vector stability and presumably prevented response. Based on this prior experience, we have redesigned our current human trials to employ a more immune-resistant MFG-based retroviral vector that is packaged in human producer cells. These trials should demonstrate whether this new generation of human cell-produced retroviral vector is more stable in the healthier patients with small volume disease. Because breast and ovarian cancers exhibit significant biologic and genetic similarities, gene therapies may be initially tested in the setting of ovarian cancer, employing peritoneal injection, and then ultimately tested in a more relevant breast cancer human disease model. Both types of patient model will be described in the following discussion.
The model system of metastatic ovarian cancer growing within a confined anatomic space (often bathed in ascitic fluid) has several advantages in safety and efficacy for studies of gene transfer into solid tumors. The pathology of metastasis into peritoneal-lined spaces often consists of small tumor implants with extravasation of tumor cells into the surrounding space, potentially allowing a reservoir for delivering gene transfer vectors to malignant cells. This pattern of spread is in contrast to other solid tumors such as breast, lung or melanoma that grow as solid three-dimensional masses anatomically located in sites that are inaccessible to currently available vector systems. Ovarian cancer provides a model system in which regional therapy (intraperitoneal infusion) could be curative in a reasonable percentage of cases. We have reported [2,36*] that the uptake and expression of the vectors can be readily assessed in this model system because direct access to the peritoneal cavity is possible through an implantable peritoneal catheter.
Because present gene therapy tools preclude systemic treatment strategies, we are initiating a regional treatment protocol to test the safety and potential efficacy of a human producer cell-derived MFG-BRCA1 gene therapy by studying the effect of BRCA1 retroviral gene transfer into breast cancer tumors that have recurred on the chest wall. The study population will consist of women who have failed one course of standard therapy and have biopsy-proved metastatic breast cancer involving the chest wall. Nodular chest wall disease constitutes a particular pattern of metastatic or recurrent breast cancer that is frequently resistant to treatment with standard therapy. We have selected chest wall recurrence of breast cancer for this study because of accessibility of the tumor both for vector administration and for biopsy and biologic analysis. Chest wall nodules will be biopsied and then injected daily for 4 days with MFG-BRCA1 viral vector. The tumors will then be excisionally biopsied 1-4 weeks later, so that molecular and cellular studies of gene transfer, expression, and immune response can be evaluated. Because these nodules are often multiple, we will inject paired nodules with a placebo retroviral vector MFG in order to test directly whether observed effects are gene related or merely nonspecific effects of retroviral vector injection in humans. Immunologic and molecular studies will be performed on blood samples and tissue samples to evaluate immune response and vector pharmacokinetics, as in the ovarian cancer trial.
Genetic correction strategies are presently being developed and tested in animal models for human malignancies and in early patient trials. The cancer susceptibility genes p53 and BRCA1 have been tested in lung cancer and ovarian cancer patients, respectively, and have shown some potential for this antitumor strategy. p53 gene therapy may be effective even against tumors that lack p53 mutations, because p53 may function as a growth inhibitor in a variety of gene transfer settings. The observation that sporadic breast and ovarian cancers show decreased BRCA1 expression indicates that BRCA1 gene therapy may be effective even against sporadic breast or ovarian cancer without BRCA1 gene mutations. Although initial approaches to human gene therapy focused on germline inherited diseases, it has become evident that somatic genetic diseases like cancer represent appropriate targets for somatic gene therapy.
The clinical application of gene correction therapy will require advances in both basic science and clinical research. Key problems at present include the degradation of vector by the immune system and a need for higher levels of gene transduction. Solutions will require the development of improved vectors, improved vector delivery systems, and the fine-tuning of human gene therapy in appropriate models of human cancer.
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