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Journal of Clinical Oncology, Vol 19, Issue 14 (July), 2001: 3422-3433
© 2001 American Society for Clinical Oncology

Cationic Liposome-Mediated E1A Gene Transfer to Human Breast and Ovarian Cancer Cells and Its Biologic Effects: A Phase I Clinical Trial

By Gabriel N. Hortobagyi, Naoto T. Ueno, Weiya Xia, Su Zhang, Judith K. Wolf, Joe B. Putnam, Paul L. Weiden, Jie S. Willey, Mary Carey, Donna L. Branham, Joy Y. Payne, Stanley D. Tucker, Chandra Bartholomeusz, Robert G. Kilbourn, Robert L. De Jager, Nour Sneige, Ruth L. Katz, Pervin Anklesaria, Nuhad K. Ibrahim, James L. Murray, Richard L. Theriault, Vicente Valero, David M. Gershenson, Michael W. Bevers, Leaf Huang, Gabriel Lopez-Berestein, Mien-Chie Hung

From the Departments of Breast Medical Oncology, Molecular and Cellular Oncology, Blood and Marrow Transplantation, Gynecological Oncology, Thoracic Surgery, Pathology, and Bioimmunotherapy, and Section of Immunobiology and Drug Carriers, The University of Texas M.D. Anderson Cancer Center, Houston; RGene Therapeutics, Inc, Woodlands, TX; Department of Internal Medicine, Medical Oncology, Rush-Presbyterian St Luke’s Medical Center, Chicago, IL; Department of Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, PA; and Section of Hematology/Oncology, Virginia Mason Medical Center, and Targeted Genetics Corp, Seattle, WA.

Address reprint requests to Gabriel N. Hortobagyi, MD, Department of Breast Medical Oncology, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd, Box 56, Houston, TX 77030; email: ghorto{at}notes.mdacc.tmc.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PURPOSE: Preclinical studies have demonstrated that the adenovirus type 5 E1A gene is associated with antitumor activities by transcriptional repression of HER-2/neu and induction of apoptosis. Indeed, E1A gene therapy is known to induce regression of HER-2/neu–overexpressing breast and ovarian cancers in nude mice. Therefore, we evaluated the feasibility of intracavitary injection of E1A gene complexed with DC-Chol cationic liposome (DCC-E1A) in patients with both HER-2/neu–overexpressing and low HER-2/neu–expressing breast and ovarian cancers in a phase I clinical trial.

PATIENTS AND METHODS: An E1A gene complexed with DCC-E1A cationic liposome was injected once a week into the thoracic or peritoneal cavity of 18 patients with advanced cancer of the breast (n = 6) or ovary (n = 12).

RESULTS: E1A gene expression in tumor cells was detected by immunohistochemical staining and reverse transcriptase–polymerase chain reaction. This E1A gene expression was accompanied by HER-2/neu downregulation, increased apoptosis, and reduced proliferation. The most common treatment-related toxicities were fever, nausea, vomiting, and/or discomfort at the injection sites.

CONCLUSION: These results argue for the feasibility of intracavitary DCC-E1A administration, provide a clear proof of preclinical concept, and warrant phase II trials to determine the antitumor activity of the E1A gene.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
LABORATORY STUDIES have shown that overexpression of HER-2/neu, also known as c-erbB2 (which encodes a 185-kd protein with tyrosine kinase activity), enhances tumorigenicity, metastasis, and resistance to chemotherapeutic agents,1-4 and clinical data indicate that patients with HER-2/neu–overexpressing breast and ovarian cancers have a poor prognosis.5,6 The adenovirus type 5 E1A gene encodes a phosphonuclear protein (transcriptional factor) that is the first viral gene product expressed in host cells after adenoviral infection. This factor in turn activates viral gene transcription and reprograms the host’s cellular gene expression to allow efficient propagation of adenovirus in the host cells.7,8 E1A has also been found to inhibit HER-2/neu expression in both rodent and human cancer cells through transcriptional repression of the HER-2/neu promoter.9-11

In this light, we previously investigated whether E1A might function as a tumor suppressor gene by repressing HER-2/neu overexpression in HER-2/neu–overexpressing cancer cells. In brief, we found that transfecting the E1A gene into genomic HER-2/neu oncogene-transformed mouse embryo fibroblast cell lines or HER-2/neu–overexpressing human ovarian cancer cell lines repressed HER-2/neu overexpression and virtually abolished the tumorigenic and metastatic potential of these cell lines.12-14 In addition, we found that the E1A gene delivered via a novel cationic liposome suppressed tumor growth and prolonged the disease-free survival of tumor-bearing mice in orthotopic models of ovarian and breast cancer.15,16 The novel cationic liposome was prepared by combining a cationic derivative of cholesterol, 3-beta-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol), with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine.17,18 Furthermore, E1A is associated with many functions that can contribute to antitumor activities, including induction of apoptosis, suppression of metastasis-related enzymes, and activation of the host immune system independent of HER-2/neu downregulation.19,20

On the basis of these results, we initiated a phase I trial of E1A gene therapy, in which the adenovirus type 5 E1A complexed with DC-Chol cationic liposome (DCC-E1A) was injected into the thoracic or peritoneal cavity of patients with breast or ovarian cancer. The goals were (1) to determine the maximum-tolerated dose (MTD) of the complex, (2) to determine if the E1A gene could be delivered into tumor cells by the DC-Chol cationic liposome gene delivery system, and (3) to evaluate the level of HER-2/neu repression as a possible marker of E1A-specific biologic activity. In addition, we evaluated tumor cells for apoptosis and cytokine expression levels that might contribute to the antitumor activity of the E1A gene.


   PATIENTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients and Work-Up
Eighteen patients with metastatic breast cancer (n = 6) or recurrent or metastatic ovarian cancer (n = 12) underwent E1A gene therapy between August 1996 and January 1998. This protocol was approved by the institutional review board of each institution involved in this trial, the National Institutes of Health Recombinant DNA Advisory Committee, and the United States Food and Drug Administration. All patients provided written, informed consent. Eligibility criteria included histologically confirmed recurrent or metastatic carcinoma of the breast or the ovary that had not experienced improvement on either conventional chemotherapy or hormonal therapy. Patients had to be at least 18 years of age, have a Zubrod performance status of less than 4, and have adequate organ function (serum creatinine < 1.5 mg/dL; total bilirubin, AST, and ALT < three times the upper limit of normal). Patients should not have received any anticancer therapy for at least 3 weeks before initiating the E1A gene therapy. Patients with breast cancer were required to have a tumor that overexpressed HER-2/neu (ie, a tumor in which more than 10% of the cells exhibited the HER-2/neu signal and in which the signal intensity was stronger than 1+).21

DCC-E1A Administration
Patients received injections of DCC-E1A complex (E1A complexed to the lipid carrier DC-Chol/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) once a week through a Tenckhoff catheter (Quinton Instruments, Seattle, WA) placed either in the pleural cavity (in breast cancer patients) or peritoneal cavity (in ovarian cancer patients). Three weekly injections followed by 1 week of rest constituted one cycle. The starting dose was 1.8 mg/m2. This dose was then escalated in 100% increments (ie, 3.6 mg/m2 was escalated to 7.2 mg/m2 in cohorts of patients).

Toxicity and Response Criteria
Preclinical toxicity studies of the E1A plasmid showed no major or minor toxicity after the delivery of a cumulative dose 40 times higher than the doses used in our trial.22 Now, as a means of defining the MTD of the DCC-E1A complex, each toxicity seen in the present trial was assessed, documented, and classified at its first appearance as related, probably related, possibly related, or unrelated to DCC-E1A administration. The MTD was defined as the highest dose at which less than two of six patients experienced more than a grade 2 drug-related toxicity. The severity of each toxicity was coded according to the National Cancer Institute’s common toxicity criteria. Dose-limiting toxicities were defined by both severity and intolerability to patients. Although gathering data on antitumor efficacy was only a secondary goal of this phase I trial, response to therapy was also evaluated after two cycles of gene therapy or when patients appeared to have undergone clinical disease progression. Each patient gave a medical history and underwent a physical examination, including a pelvic examination for patients with ovarian cancer, blood counts, sequential multiple analysis-12, chest x-ray, bone scan, chest and abdominal/pelvic computed tomography scans, and tumor marker (carcinoembryonic antigen [CEA], CA 27-29, CA-125) assays, as clinically indicated.

Disease progression was evaluated in terms of partial remission, stable disease (SD), or progression of disease (PD). Partial remission was defined as a more than 50% reduction in the sum of the products of the two greatest perpendicular diameters of each measurable lesion. SD was defined as a less than 25% increase or less than 50% reduction in the sum. PD was defined as the appearance of new lesions or a more than 25% increase in the sum. Patients were taken off the study if PD was confirmed or if they had severe grade 4 or irreversible grade 3 toxicities. All data were updated through January 22, 1998.

Immunohistochemical Staining
HER-2/neu, E1A, and Ki-67 expression levels in HER-2/neu–overexpressing cancer cells after administration of DCC-E1A were evaluated by immunohistochemical staining. Paraffin sections of tumor specimens were deparaffinized and dehydrated in a graded series of alcohol baths. They were then digested in 0.05% trypsin for 15 minutes, blocked in 0.3% H2O2 in methanol for 15 minutes, and treated with 1% (v/v) normal horse or goat serum for 30 minutes. Then, the slides were incubated for 3 hours at room temperature with HER-2/neu polyclonal antibody (Dako, Botany, Australia) diluted 1/300, with E1A M73 monoclonal antibody (Oncogene Science, Inc, Cambridge, MA) diluted 1/20, or with undiluted antihuman Ki-67 antigen monoclonal mouse antibody (Zymed Laboratories Co, San Francisco, CA). After extensive washing with phosphate-buffered saline (PBS), the slides were incubated for 30 minutes at room temperature with biotinylated goat antirabbit immunoglobulin (IgG) and biotinylated horse antimouse IgG antibody diluted 1/200 in PBS. The slides were subsequently incubated for 60 minutes at room temperature with avidin-biotin-peroxidase complex diluted 1/100 in PBS. The peroxidase-catalyzed product was visualized with 0.125% aminomethyl carbazole chromogen stock solution (Sigma Chemical Co, Milan, Italy). Between steps, the slides were rinsed for 2 minutes in PBS (pH 7.6) three times. After light counterstaining with Mayer’s hematoxylin (Sigma), the slides were dehydrated and mounted. To maintain interassay and intra-assay consistency among each batch of stained slides, negative controls (in which the primary antibody was replaced with an isotype-matched irrelevant IgG) and a positive control (a slide previously identified as having strongly staining tumor cells) were prepared. The prepared slides were then examined by light microscopy. Mean optical density (MOD) was evaluated using the SAMBA 4000 cell image analysis system (SAMBA Technology, Meylan, France).23 The MOD ratio was calculated by comparing the pretreatment MOD with the posttreatment MOD. The Ki-67 ratio was calculated by comparing the pretreatment Ki-67 with the posttreatment Ki-67.

Reverse Transcriptase–Polymerase Chain Reaction
The distribution of E1A gene expression was analyzed by reverse transcriptase–polymerase chain reaction (RT-PCR). Total RNA was extracted from homogenized autopsy specimens of patient no. 1 (approximately 100 µg from each different organ) using RNAzol (BiotecX Laboratories, Houston, TX) B reagents and the RNAzol protocol. The RNA samples were resuspended in 50 µL of distilled water. Before RT-PCR, 5 µg of total RNA from each autopsied organ was subjected to DNase I digestion with amplification-grade DNase I (2 units per reaction) (Gibco BRL, Grand Island, NY) to eliminate the chance of potentially contaminating the E1A DNA. RT reaction was carried out using a SuperScript preamplification system (Gibco). A 1/10 aliquot of new synthetic cDNA product from each RT reaction then was used for PCR amplification. The first set of PCR primers was ENLS1 (5'-CGG GAT CCC CAC CAT GCT CGA GCC TGA GCC TGA GCC CGA-3') and ENLS2 (5'-CGG AAT TCT TAT GGC CTG GGG CGT TTA-3'). The second set of primers was EN1 (5'-CGG GATCCC CAC CAT GAG ACA TAT TAT CTG CCA CG-3') and EN2 (5')-CGG AAT TCT TAC TCG AGG TCA ATC CCT TCC TGC ACC-3'). ENLS1 and ENLS2 were located in the exon 2 region of Ad5EIA, whereas EN1 and EN2 were located in the exon 1 region. Because these primers were originally designed for the purpose of cloning, each one has an unmatched sequence at the 5' end (underlined). PCR was carried out, without preheating, for 35 cycles (denaturation at 94°C for 1 minute, annealing at 58°C for 2 minutes, and primer extension at 72°C for 2 minutes).

Counting Tumor Clumps
After centrifugation of the collected intracavitary fluid, pellets were fixed in 10% formalin, buffered, embedded in paraffin, sectioned serially every 4 µm, placed on slides, and stained with hematoxylin and eosin. Each slide containing a fixed specimen was divided into nine segments by drawing lines on the bottom of the slide. The tumor clumps in each section were then counted under a microscope at x10 magnification, and the totals were added together. Three independent pathologists examined each slide. When the counts of tumor clumps on a slide differed between the independent observers, the slide was reexamined and a consensus on tumor clump number was reached. Percentage of tumor clumps was calculated by comparing the pretreatment tumor clump number with the posttreatment tumor clump number.

Terminal Deoxynucleotidyl Transferase–Mediated Deoxyuridine Triphosphate-Biotin Nick End-Labeling Assay
Apoptosis was evaluated by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) assay. Paraffin sections were deparaffinized and dehydrated in a graded series of ethanol and then washed in distilled water. The nuclei of the tissue sections were stripped from proteins by incubation with proteinase K (20 µg/mL) in 50 mmol/L Tris/HC1 buffer (pH 7.6) for 15 minutes at 37°C. Slides were then washed four times in distilled water and immersed in 1x terminal deoxynucleotidyl transferase (TDT) buffer (5x, 15 U/µL) (Gibco) for 15 minutes at room temperature. TDT (0.3 U/µL) and biotinylated dUTP 20 mmol/L in 1x TDT buffer were then added to cover the sections, which were then incubated for 1 hour at 37°C. The slides were washed with PBS six times. The sections were blocked with 10% normal horse serum for 30 minutes at room temperature and then with avidin-biotin-peroxidase complex diluted 1/100 for 1 hour. Slides were washed with PBS, then developed with 0.125% amino-ethyl carbazole buffer for 5 minutes. Finally, the sections were counterstained with Mayer’s modified hematoxylin for 1 minute. The percentage of apoptosis was calculated by counting the apoptotic cells in the entire cell population.

Cytokine Assay
Tumor necrosis factor-{alpha} (TNF-{alpha}) and interferon-{gamma} (IFN-{gamma}) levels in the supernatants of intracavitary fluid samples were measured using solid-phase sandwich enzyme-linked immunosorbent assay kits (ELISA, Cytoscreen Immunoassay Kit; Biosource International, Camarillo, CA) according to the manufacturer’s instructions, along with monoclonal antibodies specific for TNF-{alpha} and IFN-{gamma}. Standard curves were generated by serially diluting standard samples composed of defined amounts of Escherichia coli–expressed recombinant human IFN-{gamma} (hIFN-{gamma}) and TNF-{alpha} (hTNF-{alpha}). The minimum detectable dose was >= 4 pg/mL for hIFN-{gamma} and >= 1 pg/mL for hTNF-{alpha}. Biotinylated antibodies such as anti–hIFN-{gamma} and anti–hTNF-{alpha} were used as specific second antibodies. To complete the four-member antibody sandwich and allow final detection of hIFN-{gamma} and TNF-{alpha}, streptavidin-peroxidase was added and allowed to bind to the biotinylated antibody. A substrate solution was then added, which in turn was acted upon by the enzyme to produce yellowish color.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Patient and Treatment Characteristics
A total of 18 heavily pretreated women who had either breast cancer (n = 6) or ovarian cancer (n = 12) were included in this trial (Table 1). The median patient age was 55 years (range, 34 to 73 years) and the median Zubrod performance status was 2 (range, 0 to 3). All 18 patients had either metastatic or recurrent disease that had progressed after multiple treatments (surgery, chemotherapy, and/or hormonal therapy) before DCC-E1A administration. The median number of previous chemotherapy regimens per patient was three (range, one to six); in five patients, their treatment had included high-dose chemotherapy with autologous transplantation. In 12 patients (six with breast tumors and six with ovarian tumors), the tumor overexpressed HER-2/neu. To allow for biologic assays before and after administration of DCC-E1A complex, patients consented to undergo multiple samplings of ascites or pleural effusions.


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  		Table 1.  Characteristics of Patients and Effect of DCC-E1A Complex
 
Patients were continuously monitored for the appearance of toxicities and evaluated for disease response every two cycles or as clinically indicated. The treatment was continued until the appearance of either severe toxicities or disease progression in the manner defined in the Patients and Methods section. The E1A plasmid was given at three doses to consecutive cohorts of patients: 1.8 mg/m2 (n = 6 patients), 3.6 mg/m2 (n = 7 patients), and 7.2 mg/m2 (n = 5 patients). A median of six total injections (range, one to eight) were given over two cycles, and the median cumulative dose of E1A plasmid was 10.8 mg/m2 (range, 5.4 to 32.4 mg/m2).

Toxicity and Clinical Outcome
In defining the MTD of the DCC-E1A complex, any observed toxicity was classified as either related, probably related, possibly related, or unrelated to the DCC-E1A administration at the time the toxicity developed (Table 2). No patients died as a result of the treatment. However, all five patients who received the highest dose of E1A plasmid (7.2 mg/m2) developed moderate to severe nausea, vomiting, and discomfort (pain or burning) at the sites of injection. As a result, the E1A plasmid dose was reduced by 50% (3.6 mg/m2) in four of those patients and treatment terminated in one. In most patients (77.8%), self-limited fever (temperature up to 103°F) developed 3 to 48 hours after injection of the DCC-E1A complex regardless of dose. In most of those cases, however, the fever responded to acetaminophen or nonsteroidal anti-inflammatory analgesics. Thus, the MTD of the DCC-E1A complex was fixed at 3.6 mg/m2.


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  		Table 2.  Toxicities
 
Even though clinical response was not an end point in this trial, SD at the injection sites and improved performance status were noted in three patients (nos. 2, 3, and 6) after two cycles of E1A gene therapy. However, none of the three patients could proceed to a third cycle: two experienced disease progression beyond the injection sites (nos. 2 and 6) and the other patient experienced a catheter-induced infection (no. 3). Furthermore, transient decreases in levels of tumor markers (CEA, CA 27-29, or CA-125) were noted in five patients (nos. 3, 4, 10, 11, and 12). Patient no. 3 was a 39-year-old white woman who experienced recurrent metastatic breast cancer after high-dose chemotherapy and autologous transplantation. The recurrence of disease in her right thoracic cavity with accompanying pleural effusion was manifested by the presence of a nonproductive cough accompanied by pleuritic chest pain and elevated serum CEA and CA 27.29 levels. After receiving two cycles of E1A gene therapy, she showed a significant improvement in breathing, accompanied by the disappearance of the pleuritic chest pain. In addition, her elevated CEA and CA 27.29 levels in serum and CEA level in the pleural fluid returned to normal over the two cycles. However, she could not continue her treatment because her site of injection became infected 1 week before she was to begin her third treatment cycle.

Expression of E1A and Repression of HER-2/neu
To confirm the findings of preclinical animal studies,15,16 we first examined whether HER-2/neu expression levels were downregulated in HER-2/neu–overexpressing cancer cells after the administration of DCC-E1A. We were able to successfully collect intracavitary fluid containing tumor cells in a timely manner from only six of 18 patients. An immunohistochemical staining technique was used to stain HER-2/neu in serial samples of tumor cells collected from the intracavitary fluids (five pleural effusions and one ascites) of these six patients (nos. 1 to 5 and 7). Five of these patients had breast cancer, and one had ovarian cancer. The stained cells were then examined by two independent pathologists, who observed that the signal intensity of HER-2/neu decreased after treatment with the DCC-E1A complex (Fig 1A). To allay concerns about quantifying HER-2/neu downregulation in the presence of a heterogeneous collection of tumor and nontumor cells, a cell image analysis system was also used to obtain a quantitative MOD of HER-2/neu expression (ie, the signal intensity of HER-2/neu at the cell membrane of tumor cells without interference from the heterogeneous cell group).23 In our six sets of serial samples taken from patients, the MOD ratio significantly decreased (39% to 98% from original MOD) after DCC-E1A complex delivery, thus indicating a downregulation of HER-2/neu expression (Fig 1B).



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  		Fig 1. HER-2/neudownregulation after administration of DCC-E1A complex. (A). Pretreatment (day 1), 3+ HER-2/neu signal intensity. (B). After one injection of DCC-E1A complex (day 15), 2+ HER-2/neu signal intensity. (C). After four injections (day 38), HER-2/neu signal intensity. (D). After six injections, 0 negative (day 57). (E). Reduced MOD ratio after administration of DCC-E1A complex.

 
After injection of DCC-E1A complex, adjoining sections of the tumor cells were immunohistochemically stained to reveal HER-2/neu and E1A and then analyzed for the distribution of E1A gene expression in those HER-2/neu–overexpressing tumor cells. While HER-2/neu downregulation was seen in tumor cells that expressed E1A (Fig 2A), E1A gene expression was detected in both the cytoplasm and nucleus of tumor cells after just a single injection of the DCC-E1A complex. Further, the E1A signal was detected in tumor cells as well as nontumor cells, such as mesothelial cells, macrophages, and lymphocytes.



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  		Fig 2. (A and B) HER-2/neu expression on day 1 and 22 of treatment (patient no. 2); (C) E1A expression on day 22; (D) systemic expression of E1A in multiple organs after locally administering DCC-E1A complex. RT-PCR of E1Asequence in RNA extracted from multiple organ sites from patient no. 1.

 
In addition, the distribution of E1A gene expression was analyzed by using the RT-PCR with two different sets of primers (second set of primer data not shown). The RNA used for RT-PCR was extracted from autopsied multiple organ sites 2 weeks after the last injection of the DCC-E1A complex into the right thoracic cavity of patient no. 1 (the cumulative dose was 5.4 mg/m2). E1A mRNA was detected in multiple organs (ie, lung, liver, and kidney) and in metastatic tumors, but not in brain, ovary, or primary breast tumors (Fig 2D). The absence of mRNA in the ovary was consistent with the results of our previous preclinical experiments in nude mice.24

Enhanced Apoptosis and Reduced DNA Replication
After we confirmed that HER-2/neu expression was downregulated after administration of the DCC-E1A complex, we looked for any decreases in the percentage of tumor clumps in the intracavitary fluid over the course of the DCC-E1A complex administration. When pretreatment and posttreatment fluids (ascites or pleural) from six patients were compared, the percentage of tumor clumps decreased dramatically after administration of the DCC-E1A complex (Fig 3A). Because E1A itself is known to induce apoptosis and may contribute to reductions in tumor cell number, we examined apoptosis of tumor cells by conducting TUNEL assays. In all five patients examined (nos. 2 to 5 and 7), the percentage of apoptotic cells increased after administration of the DCC-E1A complex (Fig 3B, C). Furthermore, we looked for a decrease in DNA replication and proliferation in these tumor cells by examining Ki-67 expression. In all six patients (patients 1-5 and 7), the percentage of Ki-67 expression significantly decreased within 15 days after the first delivery of DCC-E1A complex (Fig 3E).



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  		Fig 3. (A). Decreased number of tumor clumps after two cycles of E1A gene therapy. (B). Increased percentage of apoptotic tumor cells after administration of DCC-E1Acomplex. (C,D). Increased percentage of apoptotic tumor cells in patient no. 2 (C, pretreatment; D, posttreatment). (E). DCC-E1A complex suppressed Ki-67 expression of tumor cells in six breast cancer patients.

 
To further address whether an immunologic mechanism might have contributed to decreased cell proliferation or increased percentage of apoptotic cells, we measured different immunologic markers. These markers included lymphocyte subsets (CD3, CD4, CD8, and CD56) and the levels of IFN-{gamma} and TNF-{alpha} in the supernatants of the intracavitary fluids over time. These supernatants originated from the same samples that were analyzed for HER-2/neu expression, apoptosis, and Ki-67 analysis. IFN-{gamma} was examined because it is known to inhibit the proliferation of tumor cells and undergo increased expression in response to injection of DNA/DC-Chol complexes; TNF-{alpha} was examined because it is known to sensitize E1A-transfected cells to apoptotic signals.25-28 Analysis revealed some increase in the levels of IFN-{gamma} after delivery of the DCC-E1A complex but no correlation between this increase and suppression of proliferation (data not shown). There was no change in the subsets of lymphocytes in the intracavitary fluid (data not shown). In contrast, the TNF-{alpha} level was significantly elevated in patients no. 2, 3, and 7 (Fig 4). Interestingly, patients no. 2 to 4 and 7 had the most significant increases in percentage of apoptotic cells and in levels of TNF-{alpha} after delivery of the DCC-E1A complex. Furthermore, patients no. 2 and 3 had clinical SD at the actual site of DCC-E1A complex injection (Table 3, Fig 4C).



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  		Fig 4. TNF-{alpha} cytokine levels in the intracavitary fluid after administration of DCC-E1A complex in six patients.

 

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  		Table 3.  Summary of Biologic Assays
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In a phase I trial of DCC-E1A administration, we defined the MTD as 3.6 mg/m2 of E1A plasmid complexed with DC-Chol cationic liposome and showed that the E1A gene can be delivered via this system to both tumor and nontumor cells. Furthermore, we showed that HER-2/neu overexpression can be downregulated in HER-2/neu–overexpressing tumor cells after the transfected E1A gene is expressed.

Because preclinical toxicity studies had shown no major or minor toxicity after the delivery of a cumulative dose 40 times higher than the equivalent dose given in our trial,22 we classified any toxicity observed in our trial as either related, probably related, possibly related, or unrelated to the DCC-E1A administration at the time the toxicity developed. In many cases, it was difficult to distinguish between symptoms caused by toxicities related to DCC-E1A administration and those from underlying disease progression. Consequently, to ensure the safety of our future studies, we included all toxicities in determining our final MTD except those toxicities that we firmly considered to be unrelated to E1A gene therapy. Within 3 hours after the initial injection, severe nausea, vomiting, and pain at the injection site occurred at the 7.2-mg/m2 dose level, and fever occurred at all dose levels; therefore, we speculate that these toxicities were due to the DCC-E1A complex rather than to E1A gene expression because the transcription and translation of the E1A gene may take more than 3 hours. Further study is required to determine if the DNA/cationic liposome complex is immunogenic or releases inflammatory cytokines such as interleukin-1, TNF-{alpha}, or interleukin-6 that can cause these side effects. However, evidence from preclinical animal studies and other studies of liposomal drug delivery indicates that the DCC cationic liposome alone does not induce these cytokines.15

A preclinical E1A gene expression study showed systemic gene expression in an animal model.14 We confirmed this finding by detecting E1A mRNA systemically after injecting the DCC-E1A complex locally (Fig 2b). In fact, this is the first documented evidence that a DNA/cationic liposome complex can survive the in vivo environment to deliver a gene to distant organs. In turn, this suggests that systemic gene delivery via intravenous injection may be feasible in nonviral cationic liposome delivery system.

As shown in preclinical models, E1A exerts its antitumor effects through a variety of mechanisms, including downregulation of HER-2/neu, induction of apoptosis, inhibition of metastasis-related enzymes, and activation of the host immunosurveillance system.19,20,28,29 For the present study, we chose to monitor the HER-2/neu–expression level as a possible marker of E1A-specific biologic activity. Consequently, we observed downregulation of HER-2/neu expression after delivery of the DCC-E1A complex at doses of 1.8 mg/m2 and 3.6 mg/m2. In addition, we wanted to determine to what extent other antitumor mechanisms (eg, apoptosis and immune activation) contributed to the antitumor activity of E1A in primary human tumor cells, and, if they did, what dose of the DCC-E1A complex was required. Consequently, we found that apoptotic cell percentages increased and that proliferation was suppressed after DCC-E1A complex administration. Moreover, TNF-{alpha} levels also increased in patients whose apoptotic cell percentage increased the most. It is worthwhile mentioning that HER-2/neu overexpression can block TNF-{alpha}–induced apoptosis via the Akt/NF-{kappa}B pathway.29 Thus, downregulation of HER-2/neu by E1A may make the antiapoptotic pathway of Akt/NF-{kappa}B inactive and contribute to TNF-{alpha}–induced apoptosis in the tumors of these patients. However, these are only correlative data from a small number of patients. Therefore, future trials should monitor multiple biologic end points after administration of the DCC-E1A complex.

The next immediate step is to determine the antitumor activity of E1A in a phase II trial, the design of which will benefit from what we learned in the phase I trial. Furthermore, E1A is known to sensitize chemotherapeutic agents like paclitaxel30-32; therefore, we are currently conducting a phase I trial which combines E1A and chemotherapeutic agents. The phase I trial was difficult to conduct and complete because (1) the patients enrolled onto it had been heavily pretreated before E1A gene therapy and had bulky disease, and (2) HER-2/neu overexpression probably contributed to more rapid disease progression; it is known to be a poor prognostic marker. These factors may have contributed to the adverse events that we observed in this trial and could affect the MTD and biologic effect of the DCC-E1A complex. Therefore, in the phase II trial, we are considering including only those patients who are left with minimum residual disease after appropriate cytoreductive measures have been taken. In this way, we will more likely obtain the high E1A-transfection efficiency that is needed to induce antitumor activity.


   ACKNOWLEDGMENTS
 
Partially supported by Targeted Genetic Corporation, the Nellie B. Connally Breast Cancer Research Foundation, National Institutes of Health grant nos. CA58880 and CA60856, and U.S. Army Research grant no. 17-94-J-4315.

We thank Jude Richard for his editorial review of the manuscript, Stacey Templin for assisting us with the cytokine assays, Andrew P. Kudelka, MD, and Ralph S. Freedman, MD, for their advice for this clinical trial, David Aboulafia, MD, for clinical care of patients, and Deb Cain for data management at Virginia Mason Medical Center. More importantly, we thank all the patients for their courage, support, and cooperation.


   NOTES
 
M.C.H. is a paid consultant of and owns stock in Targeted Genetic Corporation.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
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Submitted September 29, 2000; accepted April 2, 2001.




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