Interferon gamma

Study objectives: Intrapleural immunotherapy has shown some activity in patients with malignant mesothelioma. We conducted a multicentric pilot phase II study to evaluate the tolerance and the activity of intrapleurally infused autologous human activated macrophages (AM[PHI]) in patients with stage IA, IB, and IIA malignant pleural mesothelioma (MPM).

Design: AM[PHI] derived from in vitro monocyte culture were infused into the pleura of patients every week for 8 consecutive weeks. Each infusion was followed 3 days later by an intrapleural injection of 9 millions units of [gamma]-interferon ([gamma]-IFN) in an attempt to prolong the in vivo activation of infused AM[PHI]. Response was assessed by CT scan and thoracoscopy when possible. If the patient's disease progressed after AM[PHI] treatment, an additional treatment was left to the choice of the investigator.

Patients: Nineteen patients with histologically proven stage IA, IB, or IIA MPM were enrolled. Two patients were excluded before any AM[PHI] infusion because of complications impeding infusion. Seventeen patients were actually treated. After completion of the AM[PHI] cellular therapy, 10 patients were treated with chemotherapy as their diseases progressed.

Results: The overall response rate of patients actually treated was 14%. When including the two patients enrolled but not treated, the overall response "in intention to treat" was 11%; two patients had a partial response, with a duration of response of 30 months and 3 months, respectively. One patient, who could not be evaluated by thoracoscopy because of pleural symphysis, is still alive without any clinical or radiologic sign of disease 69 months after treatment. No major adverse effects were observed during the infusion of either AM[PHI] or [gamma]-IFN, and there was no interruption of treatment because of toxicity. However, symphysis was observed in 7 of 14 patients who received the complete treatment. The median survival of patients actually treated, including those who received chemotherapy after AM[PHI], was 29.2 months.

Conclusion: Combined infusion of AM[PHI] and [gamma]-IFN was well tolerated in patients with MPM; however, it had limited antitumor activity.

Key words: activated macrophages; intrapleural immunotherapy; malignant pleural mesothelioma

Abbreviations: AM[PHI] = autologous human activated macrophages; [gamma]-IFN = [gamma]-interferon; MPM = malignant pleural mesothelioma; WHO = World Health Organization

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Malignant pleural mesothelioma (MPM) is a rare tumor, but epidemiologic studies show a sharply rising incidence. (1) In most published reports, an association with asbestos exposure is found in 60 to 80% of the cases. (2) The prognosis of malignant mesothelioma is poor, with a median survival of 9 to 12 months and few survivors after 5 years. (3)

Classical treatments such as surgery, chemotherapy, and radiotherapy are considered to have little or no effect on the outcome of the disease and on survival. (3-6) Radical surgery is usually a pleuropneumonectomy; as a single-modality treatment, it provides no improvement of overall survival. However, trimodality using extrapleural pneumonectomy and adjuvant chemotherapy followed by radical external radiation to the hemithorax has been proposed and offers a 5-year survival of 45% for patients with limited epithelial MPM and no mediastinal involvement; however, this radical approach concerns only selected patients. (7) Radiotherapy has no value as a curative treatment, but is used on the needle tracks and scars to prevent the occurrence of parietal infiltration by tumor cells, (8) and to treat thoracic pain. Chemotherapy has been largely unsuccessful with single agents or combination regimens; the response rates are < 30%, with almost no complete response. When observed, responses are of short duration and the impact on survival is unproven. (9,10) In order to improve the survival of the patients with MPM, new therapeutic strategies based on immunotherapy or gene therapy have been developed (11,12); the intrapleural treatment seems to be appropriate in this tumor usually limited to one hemithorax. [gamma]-Interferon ([gamma]-IFN) administered by an intrapleural route induces complete responses in limited-stage diseases. (11) The mechanism underlying the antitumor action of [gamma]-IFN is complex: (1) a direct antiproliferative effect on tumor cells (13); (2) activation of pleural macrophages, (14) which became cytotoxic for the tumor cells; and (3) activation of T lymphocytes and natural killer cells by cytokines released by macrophages. These results prompted us to propose a treatment of MPM by autologous [gamma]-IFN--activated macrophages (AM[PHI]).

Indeed, given the tumoricidal potentiality of human AM[PHI] in vitro and in vivo in murine models, (15-18) we and others performed phase I studies of anticancer adoptive immunotherapy using monocyte-derived AM[PHI] as effector cells. (19-23) However, the antitumoral efficiency of this approach was limited in those patients with advanced disease. One complete response was reported in a patient with a peritoneal malignant mesothelioma treated by intraperitoneal adoptive immunotherapy with AM[PHI], (24) suggesting that local treatment could have some efficacy. The aim of the present study was to evaluate the antitumoral activity of intrapleurally infused AM[PHI] in patients with limited MPM. This trial is a noncontrolled multicentric pilot phase I/II study in 17 patients with stage IA, IB, or IIA MPM. For each patient, leukopheresis was performed every week for 8 consecutive weeks, mononuclear cells were then differentiated into macrophages by culture, and in vitro activation of macrophages to cytotoxicity was performed by [gamma]-IFN. The AM[PHI] were then purified and reinfused to the patient. Based on data showing the possible reactivation of the antitumor activity of AM[PHI], (25) each AM[PHI] infusion into patients was followed 3 days later by an intrapleural injection of 9 million units of [gamma]-IFN in an attempt to prolong in vivo activation.

MATERIALS AND METHODS

Patient Selection

Eligibility was restricted to patients with stage IA, IB, or IIA MPM as defined in Butchart modified classification. (26) Stage IA patients had a tumor limited to ipsilateral parietal pleura, stage IB patients had tumor invasion of parietal and visceral pleura, and stage IIA patients had ipsilateral seeding to the chest wall, mediastinum, or pericardium. Other inclusion criteria were as follows: (1) histologic proof of epithelial or mixed MPM; diagnosis had to be performed by standard histology, immunocytochemistry (keratin, vimentin, carcinoembryonic antigen), and confirmed by the French MPM histologic panel; (2) pleural cavity free of adherences; (3) no previous chemotherapy, curative radiotherapy, surgery, or immunotherapy; (4) performance status [is greater than or equal to] 60% according to Karnofsky scale; (5) age < 75 years; and (6) granulocyte count [is greater than or equal to] 2,000/[micro]L and platelet count [is greater than or equal to] 125,000/[micro]L. Preventive radiotherapy of the point of entry of the thoracoscope was recommended. The protocol was approved by the local ethical committee, and all patients gave written informed consent.

Pretherapeutic staging consisted of full history, clinical examination, chest radiography, and chest CT scan (with prone position to control the effusion mobility, and abdominal slices), thoracoscopy with biopsies for histologic examination (the lesions had to be precisely described and, when possible, measured during the thoracoscopy), blood counts, and standard laboratory parameters. During the study, a twice-weekly evaluation was performed before each intrapleural infusion, including physical examination, determination of changes in performance status, assessment of all toxicities observed after the previous infusion; chest radiography and routine hematologic and biochemical parameters were performed once a week.

Treatment Protocol

Isolation and Culture of Monocytes: The technique was standardized and gave identical results in a preclinical study carried out in the four producing laboratories, namely Paris-St Antoine, Paris-Immuno-Designed Molecules, Rennes, and Vandoeuvreles-Nancy. For each patient, leukopheresis was performed every week for 8 consecutive weeks. Approximately 10 x [10.sup.9] mononuclear cells (25 to 40% of monocytes) were collected by processing 1.5 to 2 masses of whole peripheral blood using a blood separator (COBE Spectra LRS Leukoreduction System; COBE BCT; Lakewood, CO). Monocytes were then differentiated into macrophages under standard operating procedures by a 7-day culture of mononuclear cells as previously described, (27) and using a specific designed device (MAK Cell Processor; Immuno-Designed Molecules; Paris, France). The device contained the following disposal: air permeable hydrophobic bags, Iscove modified Dulbecco medium supplemented with L-glutamine (2 mM), pyruvic acid (2 mM), indomethacin (5 x [10.sup.-6] mol/L), mercapthoethanol (3 x [10.sup.-5] mol/L) and nonessential amino acids (2%), endotoxin-free buffers for washing and elutriation of cells, and antibodies for quality control. Briefly, mononuclear cells were seeded at 5 x [10.sup.6] cells/mL during 7 days in culture bags at 37[degrees]C and 5% carbon dioxide, 95% humidified atmosphere in the culture medium supplemented just before the culture with granulocyte macrophage-colony stimulating factor (500 U/mL; Sandoz-Novartis; Rueil-Malmaison, France) and 2% of autologous serum, Activation of macrophages to cytotoxicity was performed by incubation in the last 18 h of the culture with 250 U/mL of [gamma]-IFN (Boehringer Ingelheim; Reims, France). On the morning of day 7, AM[PHI] were purified by elutriation (Beckman Avanti J20 centrifuge with a JE 5.0 rotor; Beckman Coulter; Miami, FL) and resuspended in 100 mL of saline solution to be further reinfused to the patient not later than 2 h after the completion of the procedure. A cell sample was taken in order to evaluate by flow cytometry the membrane phenotype of AM[PHI] with antihuman leukocyte antigen DR, CD14, and CD64 monoclonal antibodies as described elsewhere. (27)

Intrapleural Infusion of AM[PHI]: An implantable port related to a pleural catheter (BardPort Implanted Port; Bard Access Systems; Salt Lake City, UT) was implanted in the subclavicular region (Fig 1). In order to avoid the infiltration of the needle tracks and scars by tumor cells, a local irradiation (7 Gy/d during 3 days) was performed. The first infusion of AM[PHI] was administered within 10 days after its implantation. AM[PHI] were infused via the implantable port into the pleural cavity over a period of 3 h. The effusion was drained before the first AM[PHI] infusion.

[FIGURE 1 OMITTED]

Intrapleural Administration of [gamma]-IFN: A dose of 9 million units of [gamma]-IFN was injected in the pleural cavity 3 days after each AM[PHI] infusion. Eight AM[PHI] infusions and eight subsequent [gamma]-IFN infusions were performed for each patient. The treatment was delivered on an outpatient basis. Corticosteroids were strictly prohibited during the study. Paracetamol could be used in case of fever. In case of massive effusion with mediastinal shift and/or major dyspnea, a thoracocentesis with fluid removal was performed before the injection of macrophages.

AM[PHI] Bacteriological Controls: Aerobic and anaerobic bacteriological controls were performed at day 0 and day 6 of the culture, and after completion of the preparation procedure. The results were always negative and will not be reported in the chapter results.

Response and Toxicity Criteria

Response to treatment was assessed 2 weeks after completion of the treatment according to World Health Organization (WHO) criteria; lesions were measured before and after the treatment by thoracic CT scan, and a thoracoscopy was performed if there was no evidence of progression on CT scan. Complete response was defined as the disappearance of all target lesions on CT scan and thoracoscopy with negative histologic findings; a partial response was defined as a > 50% decrease in tumor size evaluated by CT scan. Objective response had to be confirmed by another CT scan measurement 4 weeks later. Additional treatment was left to the choice of the investigator. Staging and responses were assessed by an external review panel after the completion of the study. Toxicity was graded by the WHO criteria.

[sup.111]In-Labeled AM[PHI] Imaging

In order to assess the homing of AM[PHI] to the tumoral lesions, a scintigraphic study using radiolabeled AM[PHI] was undertaken in five patients. An aliquot of [10.sup.8] AM[PHI] was sampled out of the bag for radioactive labeling according to a previously published method. (28)

Image acquisition sequences were performed immediately, and at 1, 2, 3, 6, 12, and 48 h after AM[PHI] infusion. They consisted in two static thoracic views (posterior and anterior, 5 min each) and a 10-min tomographic acquisition of the thorax (64 steps). All acquisitions were performed using a rectangular, large field-of-view camera tuned to 173 keV and 247 keV energy peaks with 20% windows. Tomographic sections were reconstructed using a Wiener filter and a Sophy camera (DSV rectangular model; Sopha Medical; Buc, France). A computer interfaced with the Sophy camera. Planar views and tomographic sections were visualized on a gray scale.

Statistical Methods

In order to detect a response rate of [greater than or equal to] 20% at the 95% confidence level, the number of 14 evaluable patients was retained according to the Gehans' method. The duration of response was determined as the interval between the beginning of treatment and the progression of disease. The duration of survival was measured from the start of treatment to death or last follow-up visit. Overall survival curves were obtained using the Kaplan-Meier method.

RESULTS

Between August 1994 and June 1997, 19 patients (mean age, 60 years; range, 45 to 75 years) were entered into the study (Table 1). Two patients could not receive the treatment, one patient because of empyema secondary to catheter-related infection, and the other patient because of pleural symphysis; these two patients were not evaluable for toxicity. Seventeen patients were treated, Fourteen patients received the complete treatment (eight intrapleural injections of autologous AM[PHI] and eight intrapleural injections of [gamma]-IFN). Three patients (patients 7, 12, and 17) received three, three, and five injections of both AM[PHI] and [gamma]-IFN, respectively; the treatment was then interrupted because of rapid progression of their disease. Overall, 123 courses were administered. For the patients who completed the treatment, the cumulative doses of AM[PHI] infused ranged from 8 to 20.5 x [10.sup.9] cells (9.8 to 25.6 x [10.sup.8] cells per injection).

Toxicity

Seventeen patients were available for evaluation of toxicity (Table 2). No major adverse effects were observed during the infusion of either AM[PHI] or [gamma]-IFN, and there was no interruption of treatment because of toxicity. Fever was the most frequent toxicity, mainly after [gamma]-IFN injection (26 of 123 courses). There was no significant hematologic toxicity. However, symphysis was observed in 7 of 14 patients who received the complete treatment; therefore, evaluation thoracoscopy was impossible in four cases and incomplete in three cases. No hospitalization was needed after AMC[PHI] or [gamma]-IFN infusion, except for one patient who presented with thoracic pain after an AM[PHI] infusion. No toxicity-related deaths were observed.

Response

Among the 17 treated patients, response was evaluated by CT scan and thoracoscopy in 8 patients, and by CT scan exclusively in 9 patients (Table 3). Two patients (patients 16 and 17), who were classified stage IB by their local physicians, were reclassified stage IIB by the external expert panel after the completion of the study, and therefore were not evaluated for response because they did not fit the initial inclusion criteria. Patient 1 could not be evaluated by thoracoscopy because of symphysis, and no definitive conclusion could be drawn from CT scan examination because he had no measurable lesion on the initial CT scan. This patient showed no clinical or radiologic sign of disease 69 months after treatment. Among the 14 evaluable patients, 2 patients (patients 4 and 11) had a partial response. The overall response rate of patients actually treated was 14%. Including the two patients enrolled but not treated, the overall response "in intention to treat" was 11%. Patient 4 was evaluated by CT scan and thoracoscopy; patient 11 was evaluated by CT scan only. The duration of response was 30 months and 3 months for patient 4 and patient 11, respectively. Patient 8 remained in stable condition during the treatment time, but later progressed and died at 20 months. Progression was observed in all of the remaining patients.

After completion of the AM[PHI] cellular therapy, 10 patients were treated by chemotherapy as their diseases progressed (Table 3). Two patients (patients 13 and 15) achieved a stabilization of their disease. The disease in patient 13 again progressed after 26 months, and this patient died at 45 months. Patient 15 was still alive in December 2000, with a follow-up of 47 months. The median survival of patients actually treated, including those who received chemotherapy after AM[PHI] treatment was 29.2 months (Fig 2). At last follow-up (December 2000), 4 of the 17 patients were alive. Patient 4 had completed chemotherapy and had a very slow-progressing disease. The disease in patients 15 and 3 was progressive at 47+ and 41+ months, respectively; patient 1 was still disease-free at 69+ months.

[FIGURE 2 OMITTED]

[sup.111]In-labeled AM[PHI] Imaging

[sup.111]In-labeled AM[PHI] imaging was performed in five patients at the first AM[PHI] reinfusion. The procedure was well tolerated. In four instances, an important uptake of [sup.111]In-labeled AM[PHI] was noted into the pleura, and the [sup.111]In-labeled AM[PHI] were still present on the image acquisition sequences performed 48 h after the AM[PHI] infusions. A representative result is shown in Figure 3; a dense location of [sup.111]In-labeled AM[PHI] is seen at the pleural tumor region (seen on CT scan). For one patient, no specific uptake in the pleural space was observed, and only a diffuse labeling corresponding to the dilution of the [sup.111]In-labeled AM[PHI] in the pleural fluid was observed.

[FIGURE 3 OMITTED]

DISCUSSION

Our study is the first carried out with AM[PHI] in patients with MPM. The toxicity is mild, and it must be emphasized that all patients were treated on a outpatient basis. The fever, mainly observed after the [gamma]-IFN injections, was transient and mild (grade 2) and well controlled by paracetamol treatment. However, the emergence of frequent pleural symphysis raises concern. This was not described in trials (11,29,30) using locally infused cytokines. This symphysis might have been favored by the large amount of injected AM[PHI] and should have been avoided by a larger volume of liquid to flush. It often precluded response assessment by thoracoscopy.

The tumor response rate was lower than expected. The overall median survival of patients actually treated, including receiving chemotherapy after AM[PHI] treatment, was of 29.2 months. Although the correlation of this survival with the AM[PHI] treatment is difficult to establish, its duration is quite interesting, as compared with other first-line treatments. Nevertheless, we were aware that spontaneous prolonged survival has been reported in early-stage diseases. (26) [sup.111]In-labeled AM[PHI] imaging preformed in five patients demonstrated some homing of the AM[PHI]s to the tumor vicinity. Scintigraphic analysis performed 48 h after the infusion showed the permanence of [sup.111]In-labeled AM[PHI] in the pleura, what justified our attempt to reactivate them by an [gamma]-IFN intrapleural injection.

Conventional treatments such as surgery, radiotherapy, and chemotherapy are disappointing in patients with MPM. The reported median survival was 13.4 months for patients with stage I, and 9.4 months for patients with stage II. (26) Thus, new therapeutic strategies are now being explored. The rationale for immunotherapy is the existence of immune abnormalities in patient with MPM. (31-33) Moreover, some human mesothelioma cell lines are sensitive in vitro to cytokines like interferon-[alpha], [gamma]-IFN, and interleukin 2, (13,34,35) and to some immune cells, (36) which suggests that immunointervention would be beneficial. Intrapleural administration of interleukin 2 has resulted in objective responses in 19 to 55% of the cases in two trials (29,30) including low numbers of patients in the early stages of disease. A multicentric phase II study testing intrapleural treatment with [gamma]-IFN in patients with a stage I and II MPM induced objective responses in 20% of the cases, (11) with a duration of response of 17 to 19 months. Monti et al (37) showed that intrapleural concentration of both soluble CD8 and neopterin was increased after intrapleural infusion of [gamma]-IFN, thus demonstrating an in situ activation of CD8-positive cells and macrophages.

Intrapleural treatment has appeared of interest for patients with MPM because this tumor often remains for a long time localized in the thorax. (38) The intracavitary immunotherapy allows a high and prolonged intrapleural concentration of active drugs in contact with tumoral cells and local immune effector cells, without systemic adverse effects (29,39); this suggests that local treatment would improve the antitumor activity. In our study, the conditions were combined to expect maximum antitumor activity of AM[PHI]: (1) the patients had limited disease, which should favor response; (2) the local treatment allowed effector cells to be in close contact with tumor cells, and the large amounts of AM[PHI] infused resulted in a high effector cell/target cell ratio (these two latter conditions were demonstrated to result in better antitumoral effect in vitro; (18) and (3) combined intrapleural infusion of [gamma]-IFN was expected to prolong the duration of AM[PHI] activation.

In conclusion, local immunotherapy with AM[PHI] was well tolerated. In vivo antitumoral activity, although observed in some patients, seems limited and remains to be confirmed. However, we think that our data will be useful for future clinical trials dealing with infusion of effector cells at the vicinity of tumors. A clinical trial of intraperitoneal infusion of AM[PHI] combined with bispecific antitumor antibodies in ovarian cancer is presently in progress, and preliminary results are encouraging. Further studies are needed to determine the best modalities of treatment using these effector cells.

ACKNOWLEDGMENT: The authors thank Dr. T. Danays (Boeringher Ingleheim; Reims, France) and Dr. P. Ledru (Sandoz-Novartis; Rueil-Malmaison, France) for providing them with [gamma]-IFN and GM-CSF respectively, to Dr. E. Michel (Immuno-Designed Molecules; Pads, France) for collection of clinical data, and Dr. J. Schlusselberg (Brussels, Belgium) for statistical analysis.

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* From the Department of Pneumology, (Dr. Monnet), Center Hospitalier Intercommunal de Creteil, Creteil; the Department of Oncology (Dr. Breau), Hopital Avicenne, Bobigny; Department of Pneumology (Dr. Moro), Center Hospitalo-Universitaire, Grenoble; Department of Pneumology (Dr. Lena), Hopital Pontchaillou, Rennes; Institut Jean Godinot (Dr. Eymard), Reims; Department of Pneumology (Dr. Menard), Hopital de Brabois, Vandoeuvreles-Nancy; Service de Medecine Nucleaire (Dr. Vuillez), Center Hospitalo-Universitaire, Grenoble; Immuno-Designed Molecules (Drs. Chokri and Romet-Lemonne), Paris; and Inserm U76 (Dr. Lopez), Laboratoire de Therapie Cellulaire, Faculte de Medecine Saint Antoine, Paris, France.

Manuscript received May 24, 2001; revision accepted December 6, 2001.

Correspondence to: Manuel Lopez, MD, FCCP, Laboratoire de Therapie Cellulaire, Faculte de Medecine Saint Antoine, 27 rue de Chaligny, 75012 Paris, France; e-mail: manuel.lopez@chusa. jussieu.fr

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