Randomized Controlled Study of Adjuvant Immunotherapy with Nocardia Rubra Cell Wall Skeleton for Inoperable Lung Cancer1

[CANCER RESEARCH 43, 5575-5579, November 1983]

Randomized Controlled Study of Adjuvant Immunotherapy with Nocardia rubra Cell Wall Skeleton for Inoperable Lung Cancer1

Yuichi Yamamura,2 Takeshi Ogura,3 Mitsunori Sakatani, Fumio Hirao, Susumu Kishimoto, Masahiro Fukuoka, Minoru Takada, Masaaki Kawahara, Kiyoyuki Furuse, Osamu Kuwahara, Harumichi Ikegami, and Nobuya Ogawa

Osa/fa University Kamiyamada, Suita, Osaka 565 [Y. Y.J; Department of Internal Medicine, Osaka University Medical School, Fukushima-ku, Osaka 553 [T. O., M. S., F. H., S. K.]; Habikino Hospital, Habikino, Osaka 583 [M. F., M. T.]: Kinki Central Hospital, /Vagasene Sakai, Osaka 591 [M. K., K. F.]; Toneyama Hospital, ToneyÃ¡ma Toyonaka, Osaka 560 [0. K.]; Osaka Center for Adult Diseases, Higa$hinari-ku, Osaka 537 [H. I.]; and Department of Pharmacology, University of Ehime School of Medicine, Onsen-gun, Ehime 791-02 [N. 0.Â¡,Japan

ABSTRACT laboratories (2). Animal experiments performed by us and others suggest that N-CWS is therapeutically effective in tumor-bearing In order to evaluate the clinical benefit of Nocardia rubra cell animals, mainly through production of specific killer T-cells as wall skeleton (N-CWS), a randomized controlled study was per well as tumoricidal macrophages (8, 12, 16, 17, 19, 20, 21). A formed with inoperable lung cancer patients entered in 5 insti preliminary Phase I study showed that N-CWS can be given to tutions from October 1978 to June 1981. Patients without pleural cancer patients in an advanced stage without serious adverse effusions were treated initially with conventional therapies such reactions (23). Thus, the present randomized controlled study as chemotherapy and/or radiotherapy, according to common was undertaken in collaboration with 4 affiliated hospitals to protocol, and then patients in performance statuses 0 to 3 were examine the adjuvant therapeutic effect of N-CWS on survival of randomized into control and N-CWS groups with stratification patients with inoperable lung cancer. into 16 categories according to 4 histolÃ³gica! types and 4 clinical stages. In the N-CWS group, 400 u,g N-CWS were initially MATERIALS AND METHODS injected once or twice into the bronchial tumor using a fiberoptic bronchoscope, and subsequently 200 ng of N-CWS were in Patients. Patients with inoperable cytologically proven primary lung jected at monthly intervals into the skin from the shoulders to cancer were entered in the present trial from October 1978 to June upper arms. Of 309 patients, 118 patients in the N-CWS group 1981. The criteria for patient entry were: no history of other cancers; no and 108 patients in the control group were eligible for statistical evidence of significant dysfunction in vital organs such as heart, kidney, analysis. There was statistically no significant difference in sur lung, or liver; age younger than 76 years; and no previous treatment for vival rate between the control and the N-CWS groups. According cancer. The eligible patients were carefully graded into Stages I, II, III, and IV according to the Japan Clinical Staging System (9) which closely to histological type, significant prolongation of the survival period corresponds to the International Union Against Cancer Staging System was observed in patients with small-cell carcinoma. The 97 (7). The patients without pleural effusion were classified into 16 groups patients with pleural effusions were initially randomized into according to a combination of 4 histological types and 4 clinical stages control and N-CWS groups. In the control group, local chemo before initial conventional therapy. The patients with pleural effusion therapy with Adriamycin was performed and, in the N-CWS were treated under another protocol of local therapy. group, local administrations and monthly intracutaneous injec Biological Response Modifier. Immunological and biological charac tions of N-CWS were given. Tube thoracostomy was performed teristics of N-CWS have been reported previously (12, 16, 18, 20, 21, 23). The agent used in the present trial was a squalene-treated, freeze- in both groups. The local response rate was statistically greater in the N-CWS dried form manufactured aseptically by Fujisawa Pharmaceutical Co., Ltd. (Osaka, Japan). Informed consent for use of this agent was obtained group than in the control group, and survival period was also from all patients in the N-CWS group. prolonged significantly in the N-CWS group. The main adverse Treatment Schedule. Modality of initial conventional therapy for pa reactions to N-CWS were skin lesions in the injected sites and tients without pleural effusion was prescribed according to histological fever, but these were temporary and not serious. type and clinical stage as summarized in Table 1. In this protocol, radiotherapy for primary tumor consisting of 1000 rads/week was given generally in 6 cycles for squamous cell carcinoma and 3 cycles for INTRODUCTION localized small-cell carcinoma. Chemotherapy (CT-1) for small-cell carci Although the use of adjuvant immunotherapy has not been noma consisted mainly of cyclophosphamide (600 mg once for 4 weeks; Shionogi Pharmaceutical Co., Ltd., Osaka, Japan), 3-[(4-amino-2-methyl- extensively investigated in inoperable lung cancer, there have 5-pyrimidinyl)methyl]-1 -<2-chloroethyl)-1 -nitrosourea hydrochloride (80 to been a few reports on prolongation of survival in patients with 100 mg once for 3 to 4 weeks; Sankyo Pharmaceutical Co., Ltd., Tokyo, nonspecific immunotherapy (3, 13). Our recent studies have focused on the clinical application of N-CWS4 prepared in our Japan), and Adriamycin (40 mg once a week for 3 to 4 weeks; Kyowa Hakko Industries, Ltd., Tokyo, Japan) given in i.v. injection. In most cases, vincristine (1 mg; Shionogi Pharmaceutical Co., Ltd., Osaka, 1This study was supported by Grants for Cancer Research from the Ministry of Japan) was added i.v. once or twice a week. The main chemotherapy drugs (CT-2) for the other 3 histological types were Adriamycin (40 mg Health and Welfare and the Ministry of Education, Science, and Culture. 2 President of Osaka University Kamiyamada Suita, Osaka 565, Japan. once for 4 weeks) and methotrexate (20 mg twice a week, at 2-week 3To whom requests for reprints should be addressed. drug-free intervals; Nippon Lederle Co., Ltd., Tokyo, Japan) or vincristine ' The abbreviations used are: N-CWS, Nocardia rubra cell wall skeleton; PS, (1 mg twice a week, at 2-week drug-free intervals), all of which were performance status; i.e., transcutaneously; i.t., intratumorally; i.d., intradermal; i.e., intracutaneously; BCG, Bacillus Calmette-GuÃ©rin;ÃŒ.I.,intralesionally. given i.v. In addition, p.o. administration of Tegafur (600 mg; Taiho Received January 6,1983; accepted August 10,1983. Pharmaceutical Co., Ltd., Tokyo, Japan) was given daily to most patients

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Table 1 Treatment modality of initial conventional therapy stageHistologySmall-cell Clinical

carcinoma + CT-1 + CT-1 Squamous cell carcinoma RT RT RT (RT) + CT-2 AdenocarcinomaIRt" RTIIRT (RT) + CT-2IIICT-1 CT-2IVCT-1 CT-2

Large-cell carcinoma Same as adenocarcinoma a RT, radiation therapy; CT, chemotherapy.

Tabte2 Distribution of characteristics ol eligible patients Response to initial Histology Clinical Stage PS therapy Squa- Adeno- mous carci- Large > Unevalu- PatientsWithoutpleuraleffusionN-CWSControlTotalWith(M/F)89/2985/23174/5222/1323/1045/23NS"(<65/65-75)63/5557/51120/10623/1222/1145/23NScell333265101cell393675Ia12noma433982333164NScell3140111IIIII4 073 50% able 2356

1770 561 43 2 1848 39 4724 7134 44 3 25104 28 5 149262349NSIV36326891019<50%87 512NS1 6743NS

pleuraleffusionN-CWSControlTotalx'testSex

a Classified histologically as adenosquamous cell carcinoma. 6 NS, not significant.

with adenocarcinoma. Usually, these initial conventional therapies were criteria authorized by the Japan Lung Cancer Society (10), which were terminated after 8 weeks. The patients, who were still in PSs 0 to 3, graded corresponding to WHO recommendation (22). Adverse reactions were randomly assigned to the control or N-CWS group by key codes in in symptomatic manifestations in the N-CWS group were recorded sealed envelopes. Randomization was performed within 2 weeks after carefully, and abnormal laboratory findings between the N-CWS and completion of initial conventional therapy. Patients in the N-CWS group control groups were compared statistically. Survival period was calcu were given intratumoral injections with 400 to 600 ^g of N-CWS under lated from the start of initial therapy to December 31,1981, for patients fiberoptic bronchoscopy, as described by Hayata ef al. (5). In the patients without pleural effusion and to April 30, 1982, for patients with pleural with no evidence of intrabronchial tumor by endoscopy, N-CWS was effusion. Survival rate curves were derived from the method of Kaplan injected into the corresponding bronchial submucosal tissue by the same and Meier (11), and the difference between the control and N-CWS method as mentioned above. In the other patients with subpleurally groups was examined by a generalized Wilcoxon test (4). Patients in localized tumor, N-CWS was injected t.c. directly into the tumor. For control and N-CWS groups who died within 30 days after the end of the patients who refused the i.t. administration of N-CWS, 200 ^g of N-CWS initial therapy were excluded from statistical analysis. were given i.d. in the shoulder or upper arms at weekly and then monthly intervals. RESULTS Patients with histologically proven cancerous effusion were assigned in advance to either the N-CWS or the control group. Patients in PS 4 Characteristics of Patients. A total of 406 patients were were also included, because patients with pleural effusion occasionally recover from PS 4 by drainage of pleural effusion. Patients in the N-CWS entered in the trial. In the study with 309 patients without pleural group were treated locally with 40 mg of Adriamycin and 400 ^g of N- effusion, 118 patients in the N-CWS group and 108 patients in CWS at weekly intervals by tube thoracostomy. In the control group, the control group were eligible for analysis. The other 83 patients Adriamycin alone was injected in the same way. Patients in both groups were ineligible for evaluation, mainly because of deviations from generally received 3 to 5 local treatments, and then the patients in the criteria of patient entry and protocol of initial therapy and aggra N-CWS group were given monthly i.d. injections of 200 ^9 N-CWS into vation into PS 4 after the initial therapy. In the study with 97 the shoulder or upper arm. patients with pleural effusion, 35 patients in the N-CWS group As the maintenance conventional therapy, most of the patients re and 33 patients in the control group were also eligible for ceived chemotherapy, with slight modifications. analysis. Characteristics of all of the eligible patients are sum Clinical and Statistical Evaluation. Eligibility of every patient entered marized in Table 2. Distribution of these characteristics was not in the study was reviewed carefully and determined by senior investiga significantly different between the N-CWS and control groups. tors from each institution and an independent controller (biostatistician). Regarding the route of administration of N-CWS, 16 of 118 Distribution of the characteristics of eligible patients was tested statisti cally between the control and N-CWS groups by x2 analysis. Response patients in the N-CWS group and 3 of 33 small-cell cancer to initial conventional therapy was evaluated as greater than 50% regres patients refused the i.t. injection through fiber optic broncho- sion of all measurable diseases as judged by more than 2 investigators. scope and received only i.e. injections. Local response to pleural effusion was also evaluated according to Survival. Survival rates of all eligible patients without pleural

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1983 American Association for Cancer Research. Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1983 American Association for Cancer Research. Y. Yamamura et al. was fever, which was usually below 39Â°and subsided within 3 because survival was prolonged in both younger and older days. Injection i.d. produced various types of skin lesions at the patients treated with N-CWS. Another characteristic to be con injection site. However, most of them were not serious, and sidered is the response to the initial conventional therapy. This there was no fever. Fever was seen in about half the patients was similar in both N-CWS and control groups as shown in Table with pleural effusion after intrapleural injection but was uncom 4. Further analysis of the survival period of patients, according mon after i.d. injection as maintenance therapy. to the response, demonstrated that survival periods in the N- Abnormal laboratory data did not differ significantly between CWS group were longer than those in the control group regard the N-CWS and control groups. However, transiently higher less of responses to the initial therapy (data not shown). serum glutamic oxaloacetic transaminase, serum glutamic pyr- In the study of patients with cancerous pleurisy, we observed uvic transaminase, serum glutamyl transpeptidase, and/or alka definite therapeutic effects of N-CWS on prolongation of survival line phosphatase were observed in fewer than 6 patients after period as well as suppression of pleural effusion, which might i.t. N-CWS injection and in 10 to 20 patients after intrapleural N- contribute to the former effect. Although no certain mechanism CWS injection. responsible for the clinical effects of N-CWS was demonstrated here, there have been some reports on immune response of cancer patients treated with N-CWS (25) and with BCG cell wall DISCUSSION skeleton, which is similar in immunological activity to N-CWS In the study of inoperable lung cancer without pleural effusion, (18). Furthermore, experimental immunotherapy with N-CWS has overall analysis revealed no significant difference in patient sur provided evidence that macrophage activation and induction of vival period between the control and N-CWS groups. According killer T-cells are mainly involved in the therapeutic effect, espe to histological type, however, significant prolongation of survival cially when N-CWS is given Â¡.I.(12, 16, 17, 19, 20). These period was observed for small-cell carcinomas. A further statis experimental results and previous clinical experience with BCG tical analysis of characteristics of small-cell cancer patients cell wall skeleton (18, 24) were of great use in establishing the showed that the N-CWS group consisted of more young patients protocol of N-CWS immunotherapy. than were in the control group, although the difference was not There have been some conflicting reports on the clinical effi significant (0.05 < p < 0.10). Therefore, we compared the cacy of adjuvant immunotherapy for inoperable lung cancer. In survival periods of the younger patients (<65 years) with those the case of small-cell carcinomas, the Southwest Oncology of older patients (a65 years). Our conclusion was that the Group (13) reported that BCG therapy demonstrated a slight, prolongation of survival period of patients in the N-CWS group yet significant, survival benefit in responders surviving more than was not the effect of the uneven age distribution of patients, 1 year but also that BCG immunotherapy had no beneficial effect on survival period or response rate (14). Cohen er al. (3) reported the significant prolongation of survival period by chemotherapy N-CWS 35 and thymosin treatment. On the contrary, Ainsner and Wiemik (1) and Holoye ef al. (6) reported no clinical benefit after che- Control 33 moimmunotherapy with methanol-extracted residue of BCG and BCG, respectively. In these trials, immunotherapeutic agents administered before, simultaneously with, or subsequently to conventional therapy were given irrespective of tumor site. In light of previous reports that a bacterial immunoadjuvant pro duced therapeutic effects mainly when given Â¡.I.,the clinical effects evidenced in the present study may be related to the suitable application of N-CWS into the tumor sites, intrabronchial tumor, and affected pleural cavity. Recently, Millar ef al. (15) also reported successful management of intrabronchial tumor by LI. injection of BCG. The effect of the N-CWS injection route on 60 120 180 240 300 360 420 480 540 patient survival is of great interest. In the present trial, however, SURVIVAL PERIOD (DAYS) only a small number of patients received the i.d. injections alone, Chart 4. Survival rates of patients with pleural effusions. and most of the patients received both i.t. and i.d. injection.

Tables Adverse reactions in N-CWStherapy

reactionAbscesscutaneous reactionLocal Route of of administration108 adverseUlcer46/105(43.8)Â° reaction Induration18(15.8)Swelling Rubor pain1 enopathy1 thema1 patients without pleural effusion Intratumoral (1.0) (43.8) Intracutaneous 13(11.4)24/3573/114(64.0) 1(0.9) 20(17.5) 18(15.8) 1(0.9)1 8(7.0)24 (0.9)Exan (0.9) 35 patients with pleural effusion Intrapleural (68.6) (2.9) (68.6) IntracutaneousIncidence 17/31 (54.8) 10(32.3)Local 3 (9.7) 1 (3.2) 2 (6.5)Systemic1 (3.2)Fever462 (6.5)Lymphad- 1 (3.2) " Numbers in parentheses, percentage in incidence.

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Thus, statistical evaluation of clinical benefit according to the mice bearing syngeneic sarcoma. Cancer Res., 47: 660-666,1981. route of administration of N-CWS was not performed. In view of 13. McCracken, J. D., Chen, T., White, J., Samson, M., Stephens, R., Coltman, C. A., Jr., Saiki, J., Lane, M., Bonnet, J., and McGavran, M. Combination the absence of severe adverse reactions, such as seen in BCG chemotherapy, radiotherapy, and BCG immunotherapy in limited small-cell immunotherapy, i.l. injection of N-CWS seems to be the prefer carcinoma of the lung. A Southwest Oncology Group Study. Cancer (Phila.), 49: 2252-2258, 1982. able adjuvant immunotherapy for inoperable lung cancer. 14. McCracken, J. D., Heilbrun, L., White, J., Reed, R., Samson, J., Seiers, J., Stephens, R., Stuckey, W. J., Bickers, J., and Livingston, R. Combination chemotherapy, radiotherapy, and BCG immunotherapy in extensive (meta static) small cell carcinoma of the lung. Cancer (Phila.) 46: 2335-2340,1980. 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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1983 American Association for Cancer Research. Randomized Controlled Study of Adjuvant Immunotherapy with Nocardia rubra Cell Wall Skeleton for Inoperable Lung Cancer

Yuichi Yamamura, Takeshi Ogura, Mitsunori Sakatani, et al.

Cancer Res 1983;43:5575-5579.

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