Ann Thorac Surg 2000;70:407-411
© 2000 The Society of Thoracic Surgeons
Original articles: general thoracic
Gene-modified PA1-STK cells home to tumor sites in patients with malignant pleural mesothelioma
Lynn H. Harrison, Jr, MDa,
Paul O. Schwarzenberger, MDa,
Patrick S. Byrne, PhDa,
Aizen J. Marrogi, MDa,
Jay K. Kolls, MDa,
Kevin E. McCarthy, MDa
a Department of Surgery, Louisiana State University Health Sciences Center, New Orleans, Louisiana, USA
Address reprint requests to Dr Harrison, Department of Surgery, Louisiana State University Health Sciences Center, 1542 Tulane Ave, New Orleans, LA 70112-2822
e-mail: lharri{at}lsumc.edu
Presented at the Forty-sixth Annual Meeting of the Southern Thoracic Surgical Association, San Juan, Puerto Rico, Nov 4–6, 1999.
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Abstract
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Background. Malignant mesothelioma is an uncommon but lethal cancer of increasing incidence, particularly among patients with a history of exposure to asbestos. Although numerous treatments have been employed, including chemotherapy, radiation therapy, surgical resection, and combinations of the above, no satisfactory treatment yet exists, and affected patients will die of this disease, usually within 12 months. Gene-based therapies constitute a new approach that offers hope of improved control of these tumors while being associated with less morbidity than conventional chemotherapeutic or surgical regimens. We demonstrated that PA1-STK cells home in vivo to mesothelioma deposits, a phenomenon that is required for optimal exertion of this therapeutic concept.
Methods. Gene-modified ovarian cancer cells expressing the thymidine-kinase gene (PA1-STK) were radiolabeled with 99Tc and infused into the pleural space of 4 patients with malignant pleural mesothelioma, then scanned to determine distribution of the cells.
Results. PA1-STK cells recognized and adhered preferentially to mesothelioma lining the chest wall.
Conclusions. Cell-based "suicide gene" therapy utilizing the "bystander effect" with the gene-modified ovarian cancer cell line PA1-STK is feasible in human pleural mesothelioma. We have shown that this trafficking and homing of the therapeutic cells to the intrapleural tumor sites, a requirement for success with this novel therapeutic concept, is also valid in humans.
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Introduction
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Malignant pleural mesothelioma remains a devastating cancer for which there is no effective cure. Over 2,000 new cases are diagnosed in the United States each year, and even patients discovered in the early stages of disease have a median life expectancy of less than 1 year [1]. Increasingly invasive procedures have produced a few long-term survivors, though at the cost of considerable morbidity and without increasing median survival in most series. In light of this, we have embarked on a new approach employing gene therapy that utilizes a novel delivery system designed to attack tumor cells directly while having minimal impact on quality of life and very low procedure-related morbidity. The basis of this approach, termed suicide gene therapy, is an alteration of the tumor genotype in such a manner that the tumor cells then become sensitive to a specific chemotherapeutic agent. The most commonly used agent in this approach uses the herpes simplex virus gene, which expresses thymidine kinase (HSV-tk). HSV-tk can be introduced into tumor cells with recombinant virus-based gene transfer vectors.
This gene therapy-based approach takes advantage of the basic mechanism used to treat herpes simplex infections with the antiviral drug ganciclovir. Although ganciclovir is phosphorylated by endogenous kinases in normal tissue, this process is substantially amplified by the HSV-tk enzyme, which converts ganciclovir into a toxic product. Thus, tumor cells expressing the HSV-tk gene are made sensitive to ganciclovir and will be eliminated.
A shortcoming of the suicide gene approach has been the inability to alter the genome of the entire population of cells within a tumor due to current limitations in gene transfer technology. It has recently been observed, however, that tumor regression will occur even if only a fraction of the tumor cells expressed the HSV-tk gene, a phenomenon that has been called the "bystander effect" [2]. A second phenomenon that has been observed and is important to this approach is the "homing" phenomenon, whereby tumor cells of a different type, when exposed to cells of the primary tumor, associate with and adhere to the primary tumor cells. Therefore, if cell lines that stably express the HSV-tk gene can be delivered to the mesothelioma cells, administration of gancyclovir will result not only in the death of the gene-modified delivery cells, but mesothelioma cells as well by virtue of the bystander effect. This concept has been applied to ovarian cancer in the clinical setting of recurrent disease that has failed cis-platin and taxol therapy with demonstrated improvement but mild to moderate toxicity in 4 of 18 patients, 3 of which experienced complete remissions [3]. Our group has demonstrated antitumor efficacy in a murine peritoneal mesothelioma model employing the HSV-tk gene-modified ovarian cancer cell line PA1-STK [4]. These cells were infused into the peritoneum of mice burdened with peritoneal mesothelioma, and significantly improved survival was demonstrated over control in those mice receiving PA1-STK cells followed by systemic gancyclovir.
On the basis of this theoretical construct and supported by the preliminary experimental and clinical studies noted above, we have undertaken a phase I clinical trial to examine the impact of a gene-based approach to the treatment of malignant pleural mesothelioma. Patients with confirmed disease underwent intrapleural instillation of HSV-tk gene-modified ovarian cancer cells in escalating doses, followed by systemic gancyclovir therapy. To prove that the basic requirement for exerting the bystander effect was also valid in humans, a portion of the infused cells were radioactively labeled with 99Tc, and their distribution was imaged by radionuclide scans. With this report, we demonstrate for the first time in humans that radioactively tagged PA1-STK cells adhere preferentially to intrapleural mesothelioma deposits, and are retained for at least 24 hours in the chest cavity, thus enabling the bystander effect to occur.
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Material and methods
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As patient safety was a principal concern in this phase I trial, rigid quality assurance and safety testing was performed to assure that PA1-STK cells were incapable of replication or prolonged survival and that the retrovirus was likewise replication incompetent. In addition, all institutional, state and federal regulations regarding informed consent and peer review were met. The details of our protocols for safety testing as well as a more extensive description of our protocol and patient selection criteria has been published previously [5]. The present study additionally examined cell labeling and homing.
Patients accepted into the Trial were admitted to the Combined LSU-Tulane General Clinical Research Center at Charity Hospital for the first 8 days of each cycle. They underwent placement of an 8F or 10F pigtail catheter into the pleural space on the affected side and then received an intrapleural infusion of cells followed by twice-daily intravenous injections of gancyclovir (5 mg/kg). The numbers of cells infused in the dose-escalation phase of the study are illustrated in Table 1. During treatment, samples of pleural fluid were obtained for analysis of cytokine profile changes of both the protein and messenger RNA using enzyme-linked immune-specific absorbent assays and reverse transcriptase polymerase chain reaction, respectively.
In selected patients, technetium-labeled PA1-STK cells were infused into the pleural space in an attempt to determine whether the cells truly "homed" to the tumor or were evenly distributed in the pleural fluid. Briefly, using a red cell tagging kit (Amersham, Piscataway, NJ), 108 cells were removed from the suspension and placed in a 15.0-mL polypropylene tube and subjected to centrifugation at 500g for 10 minutes at 4°C. The supernatant was discarded and the cell pellet was resuspended in sterile 0.9% saline and held on ice. The cells were mixed with 3.0 mL stannous chloride/pyrophosphate solution dissolved in sterile saline for 25 minutes at room temperature with gentle agitation. The 99Tc (55 mCi) label was then added to the cell suspension, the tube placed in a protected lead cylinder, and mixed with gentle agitation for an additional 25 minutes. The cell pellet was collected by centrifugation at 500g for 10 minutes at room temperature and the radioactive supernatant was collected in an appropriate waste container. The cell pellet was washed twice more in sterile saline. After the second washing step, the tagged cells were resuspended in sterile saline, and both cells and waste constituted by washing fluid were counted in a well counter. Labeling efficiency was determined by dividing the final amount of radioactivity in the cell suspension by the initial starting dose, and validated that more than 90% of the unlabeled 99Tc was present in the waste fraction. We now achieve 40% 99Tc incorporation in our tagging experiments. Radiolabeled cells were then infused into the patient’s pleural space through the indwelling pleural catheter. The patient was instructed to turn into lateral decubitus positions and supine and prone positions to assist even distribution of cells in the pleural space. Patients were scanned immediately (1 to 4 hours) after the cells were placed, with delayed imaging performed 24 hours after cell placement in all the patients and 48 hours in 1. Patients were not routinely imaged beyond 24 hours due to loss of radioactivity secondary to decay of this relatively short half-life isotope (6 hours). Planar spot acquisitions of the chest and abdomen or whole-body images were obtained using a Siemens (Erlangen, Germany) triple-headed camera. At least one set of SPECT images of the chest was performed for each patient, usually at 24 hours. Images were reconstructed in the transverse, sagittal, and coronal planes.
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Results
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Cell homing as evaluated by radionuclide imaging
Anterior and posterior static images of the chest were obtained in 4 patients 24 hours after instillation of radiolabeled PA1-STK cells into the pleural space and in 1 patient at 48 hours. These scans consistently showed localization of radiolabeled cells along surfaces corresponding to tumor deposits as indicated by computed tomography scan (Fig 1). At least one set of SPECT images of the chest was performed for each patient, usually at 24 hours, and these also confirmed localization of labeled cells in areas of known tumor involvement with absence of radiolabeled cells in free pleural fluid bathing these areas of tumor deposition (Fig 1).
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Fig 1. (A) Computed tomography scan of the chest shows a pleural-based soft tissue mass involving the lateral and superior left chest wall in a circumferential fashion. Posterior solid arrowpoints to the soft tissue mass, which has a Hounsfield unit density averaging 34 (30 to 35), whereas the open arrowpoints to a large effusion that has a Hounsfield unit density averaging 12 (10 to 15). (B) Anterior static image of the chest 24 hours after placement of 99technetium-m-labeled cells into the left pleural space. Cells have localized predominantly to the upper half of the chest with some intense focal areas in the pleural mesothelioma. Solid dark arrows point to the level of the diaphragm and larger open arrows to the soft tissue nodules. (C) Posterior static image of the chest 24 hours after placement of 99technetium-m labeled cells into the left pleural space. Cells again shown to accumulate in the tumor. Solid arrowsindicate the level of the diaphragm and open arrowspoint to the soft tissue nodules. (D) SPECT image of the chest in the coronal projection 24 hours after placement of labeled cells in the left pleural cavity. Localization again seen laterally and superiorly corresponding to the known areas of the pleural mesothelioma.
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Morbidity
No patient included in the trial experienced any morbidity attributable to the treatment. In one patient, it was not possible to complete the full dose escalation protocol because of fusion of the pleural space during the interval between treatments. Three patients required reinsertion of their pleural catheters because of catheter malfunction or dislodgment. No bleeding or septic episodes were observed.
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Comment
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Although patients with pleural mesothelioma have been subjected to a wide array of treatments that have found some success in the management of other soft tissue neoplasms, including multiagent chemotherapy [6], radiation therapy [7], photodynamic therapy [8], and attempts at surgical extirpation [9], in some series, none of these treatments has prolonged median survival. Gene-based therapy using the concept of the "suicide gene" offers the possibility of attacking tumor cells directly in a manner that has the potential of differentiating tumor cells from normal cells and concentrating the tumoricidal activity on the neoplasm with minimal impact on non-tumor-bearing tissues. In this phase I clinical trial, irradiated ovarian cancer cells transduced to express the thymidine kinase gene (HSV-tk) were labeled with 99Tc and infused into the pleural space of 4 patients with pleural mesothelioma. Technetium scanning 24 hours later confirmed what had previously been theorized on the basis of animal experiments, but that had not previously been documented clinically: the PA1-STK cells did in fact "home" to the mesothelioma cells and bind to them in some fashion, rather than remaining suspended in pleural fluid, where their contact with tumor cells would be random and less concentrated, thus undermining the tumoricidal potential of the bystander effect. The bystander effect is thought to result from the passage of toxic metabolites (in this case, the tri-phosphorylated metabolite of ganciclovir) via gap junctions established when gene-modified PA1-STK cells adhere to mesothelioma cells. A potential shortcoming of this form of therapy lies in the fact that only tumor cells near the pleural surface are likely to bind infused cells carrying the HSV-tk gene. The fibrotic nature of mesothelioma may also limit the delivery of PA1-STK cells to large segments of the tumor. Clearly, this limitation would have greatest impact in advanced disease and would be less likely to exert an influence in early disease. An additional limitation of this approach is the necessity of a free pleural space to allow exposure of the gene-modified cells to as much of the tumor surface as possible. Patients with later stages of disease frequently will have fused their pleural spaces, eliminating the possibility of delivering the PA1-STK cells to their target.
A potential though as yet undefined strength of gene-based therapy for this tumor is suggested by the fact that in vivo studies employing the bystander effect have shown a level of tumor regression that exceeds that which would be anticipated on the basis of in vitro studies. A significant body of experimental work suggests that this may be explained by upregulation of immune mechanisms, and preliminary data from our trial confirm these observations (data not shown) [2, 4, 10, 11].
Future directions for this avenue of investigation point to a systematic analysis of cytokine and cellular immune response to this therapy. It is likely that current limitations in gene vector technology will relegate gene therapy to an adjunctive role in the management of this tumor, but one that may prove helpful both in the treatment of early-stage disease and in furthering our understanding of the role the immune system plays in this and other cancers. For example, this approach may prove quite useful in limiting the complications of pleural or peritoneal spread of other cancers, even though eradication of the disease is not possible by these means. Often, promising results from animal models cannot be duplicated when translated into clinical settings. With this study, we have sought to demonstrate that the fundamental conditions requisite to this novel therapeutic concept and that have been observed in animal models of mesothelioma were also valid in human mesothelioma. Because the bystander effect comprises the basis of this form of therapy, attachment of PA1-STK cells to tumor is a critical condition supporting the potential efficacy of this approach. Another condition upon which the success of this strategy depends is the attainment of therapeutic levels of gancyclovir in the target tissue. Preliminary pharmacokinetic studies suggest that concentrations of gancyclovir sufficient to effect cytotoxicity were achieved in the pleural fluid of these patients (data not shown). A future issue will involve determination of the actual number of adhering cells and characterization of any differences in adherence to the various cell types of mesothelioma.
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Acknowledgments
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We thank Dr Scott M. Freeman (Kenilworth, NJ) for generously providing the PA1-STK cell line.
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References
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Abstract
Introduction
