No. 10]

41.

Proc. Japan

Acad.,

70, Ser. B (1994)

205

Inter feron-y as a Marker for the Effective Cancer Immunotherapy
with BCG-Cell Wall SkeletonDepartment

By Akira
HAYASHI*)of Planning and Research, Osaka Medical Center for Maternaland Child Health, 840 Murodo-cho, Izumi, Osaka 590-02(Communicatedby Kumao ToYOSHIMA,M.J. A., Dec. 12, 1994)

Abstract:Immunotherapy
with BCG-CWS (the cell wall skeleton from BacilleCalmette-Guerin)alone has been tried on the patients with various kinds of cancer, after
surgical operation and/or chemotherapy.
After the inoculation most of the patients
revealed biological responses: skin reactions (erythema, inouration and ulcer formation),
physical signs, and cytokine induction in peripheral blood (G-CSF, IL-6 and IFN-y).
Among the patients, those who revealed distinct IFN-y induction and/or strong skinreaction are alive in healthy state including a few cases of complete recovery. Those who
showed no IFN-y induction, even if they had weak skin reaction, died in a short period.
Most of them were in terminal stage or had had repeated chemical or radiation therapy
before immunotherapy.
These results suggest that nonspecific Immunotherapy
with BCG-CWS, if applied
independently of any other therapy, will provide an extremely effective way to prevent
relapse or occurrence of secondary cancer, and those who produce IFN-y responding
BCG-CWS inoculation can be regarded as perfect candidates for cancer immunotherapy
under discussion.
Key words:

Nonspecific Immunotherapy;

BCG-CWS; interferon-y;antitumor

effect.

Specific and nonspecific activation of the host immune system to reject cancer is anappealing idea. Although many researchers have challenged to this theme, it does notnecessarily follow that Immunotherapy is accepted as one of the important methods forcancer treatment. In Japan, the nonspecific Immunotherapy for cancer using BCG-CWS(the cell wall skeleton derived from Bacille Calmette-Guerin) was widely examined by
Yamamura et al. in 1970's. In their randomized clinical trial, BCG-CWS was mostly usedas supplementary to conventional therapies, and it failed to bring about good results./)
The author assumed the following two questions about the unsatisfactory results of
their clinical trial;
1. Was the clinical trial performed in proper way to give the real value of theImmunotherapy?
2. Were the patients in the trial really qualified for the immunotherapy with BCG-CWS?
If they were not, how could we decide well-qualified patients?
In order to answer these questions, the Immunotherapy with BCG-CWS was tried on a
new idea.
BCG-CWS2~was kindly supplied by Dr. I. Azuma (Institute of Immunological Science,
Hokkaido University). The preparation of oil-attached BCG-CWS was performed aspreviously reported,2~ except for the ratio of mineral oil to BCG-CWS and theconcentration of Tween 80 in saline: Drakeol 6VR, Pennsylvania Refining Co., Butler,
U.S.A. was added, 2 drops per mg, to BCG-CWS powder, and the oil-attached powder was*)Present

1-3-3 Nakamichi,

address:

Division

Higashinari-ku,of Cancer
Osaka

Immunotherapy

537, Japan.

, Centerfor Adult

Diseases,

Osaka,

i206

A.

[Vol. 70(B),HAYASHI

suspended in physiological saline containing 1.1% Tween 80. After sterillized by heating 30
minutes at 60°C, the suspension was inoculated intradermally on the external skin of upperarm according to the schedule which was composed of two phases; in the sensitization
phase, 200 µg of BCG-CWS was inoculated weekly and, usually at 3 or 4 inoculations,patients' skin reactions (erythema, inouration and ulcer formation) would reach to themaximum. Then the phase moved to the continuing phase, in which most of the patientswere inoculated 100 ug of BCG-CWS monthly. Cytokines in serum were assayed by
ELISA. IL-i (a and /3), IL-2, TNF-a, G-CSF, IFN-a and -y were determined with assay
kit from Otsuka Assay Co., and IL-6 and IL-8 were determined with the one from R & D
Co.. Among these cytokines, only G-CSF, IL-6 and IFN-y could be detected in peripheral
blood after BCG-CWS inoculation.

Fig.

1.

Clinicalcourseof a patientwithcolorectal

cancer.

op
surgicaloperation.

5-Fu

BCG-CWS
fluorouracil.

inoculation

Ara-C
MMC

cytosinearabinoside.
mitomycin-c.

andthenumbers

denotegivendosein gig.

In regard to the first question, there was a strong doubt about the program of the trial
in that CWS was mostly used with various kinds of anticancer therapy which weresupposed to impair our immune surveillance system. So the decision was made to use CWS
on the patients independent of conventional therapies, just after initial operation orchemotherapy, if possible. Starting in 1974, an independent use of BCG-CWS was tried forthe treatment of three patients, two with acute myelogenous leukemia (case 11 and 12 inNo. 10]

CancerTableI.

CancerImmunotherapy

and207

Interferon-ypatients,
their clinical and biological recordsof Immunotherapy
with BCG-CWS aloneand evaluation

Table I) and one with colorectal cancer, after obtaining their consent.
Fig. 1 shows an early part of the clinical course of the patient with relapsed
cancer accompanied by peritonitis
carcinomatosa.
The patient first complainedcolorectalof severe

pain in abdomen like ileus in September
1975, at the age of 72. After being diagnosed ascolorectal cancer, she had a surgical operation at Toyohashi City Hospital, Toyohashi,

208

A.

HAYASHI[Vol. 70(B),Japan. After the operation, as shown in Fig. 1, she was treated with conventional
combined chemotherapy (fluorouracil, cytosine arabinoside, and mitomycin-c) and her
clinical course was followed by serum carcinoembryonic antigen (CEA) level. Though she
was doing well with CEA level decreasing after the operation, her clinical course wasreversed 2 months later with CEA level beginning to increase. Further she complained offull feeling in abdomen and anorexia, and the sign of ascites appeared. Laboratory
examination disclosed hypoproteinemia and the presence of peritonitis carcinomatosa. So
the chemotherapy was discontinued and immunotherapy with BCG-CWS alone wasinitiated. After 5 months her ascites disappeared and serum CEA level decreased to thenormal. After that she completely recovered and her CEA level was maintained within
normal range until her death. Immunotherapy continued about 8 years and she completed
the natural span of life at the age of 87. Other two patients, one of them had already had arelapse at the start of immunotherapy, completely recovered by the immunotherapy withBCG-CWS alone and are still alive having no secondary cancer. These data are theexamples to show that some cancer patients could be cured, even if they had a relapse, byBCG-CWS alone.
In order to examine the second question, recently analyses were made on various
kinds of biological responses induced by the immunotherapy with BCG-CWS. Twelve
patients with 4 lung, 2 colorectal, 1 pancreatic, 1 uterine cervical, 1 gastric and 1 ovariancancer and 2 myelogenous leukemia, of whom more than half (7/12) were at stage III to IV,
were treated with intradermal inoculation of BCG-CWS alone, 100 or 200 µg monthly(Table I). The informed consent in writing was obtained from these patients. After theinoculation, most of the patients revealed some biological responses: skin reaction, lowgrade fever, alteration of laboratory indices (white cell counts and its differentials, andC-reactive protein), and cytokine induction in peripheral blood (G-CSF, IL-6 and IFN-y).
Among these biological responses, IFN-y induction and skin reaction are mainly relatedwith antitumor activity: as shown in Table I, of these 12 cases, 7 patients showed a distinct
IFN-y induction (more than 15.0 pg/ml) and/or strong skin reaction, and most of them (6/7)
are alive in healthy state including two cases of complete recovery. Those who showed noIFN-y induction (5/12), even if they had weak skin reaction, died in a short period. Most ofthem were in terminal stage or had had repeated chemical or radiation therapy before
immunotherapy. So, it is recommended that, if the patients do not reveal any expectedbiological responses including IFN-y at the first inoculation in continuing phase,
immunotherapy may be replaced by original or other conventional therapies. Case 4
showed a marked induction of IFN-y initially, but ceased to show it 6 months later after
the x-ray irradiation for neck bone metastasis, and died after 1 year.
As for the precise mechanism of the immunotherapy with BCG-CWS, there are many
unsolved problems. In the present study, it was found that BCG-CWS induced IFN-y,
especially in patients who showed good prognosis. In contrast, all patients who could not
induce it were dead. This fact might have some relation with the data that T-lymphocytefrom 13 of 14 AIDS patients completely failed to secrete IFN-y upon stimulation withspecific microbial antigen.3~ So IFN-y seemed to be one of the most important cytokines inimmune regulation4~ essential for the antitumor effect.5~ Though Yamamura's group had
tried cancer immunotherapy using BCG-CWSI~ or Nocardia-CWS,6~ this therapy finally
failed to get the approval of the Ministry of Health and Welfare of the Japanese
Government. The most important reason of this failure may have been in the inadequate
selection of the subjects; in their trial, the study patient group perhaps included many
unresponsive subjects to CWS who were having combined chemotherapy, radiation
therapy or were in far advanced stage of cancer. Most of these patients might be in theworst immunological condition for cancer immunotherapy. In order to evaluate the value of

No. 10]CancerImmunotherapy

andInterferon-y209

cancer Immunotherapy correctly, immunotherapy, as suggested in the present study,
should be performed independently of other conventional therapies and on the patients
who produce IFN-y responding BCG-CWS inoculation. Since chemotherapy has still been
regarded as the most powerful tool for cancer treatment in Japan, or perhaps in othercountries, too, most patients are given anticancer drug just after initial operation. As theindependent nonspecific immunotherapy under discussion grows in reliability withincreased number of cases, it will be approved, in near future, as the most important andnatural maintenance therapy for cancer patients.
Acknowledgements. The author is most grateful to Dr. I. Azuma (HokkaidoUniversity) for his kind supply of BCG-CWS and proper suggestion to the preparation ofthe inoculant, Dr. A. Noda (Otsuka Assay Co.) for measuring cytokines, and Drs. K.
Toyoshima, M.J. A., (Center for Adult Diseases, Osaka), K. Sekiguchi, Y. Wada and K.
Matsumoto (Osaka Medical Center for Maternal and Child Health) for their valuable
discussion and encouragement.
References
1) Yamamura, Y. et al. (1976): Immunotherapy of cancer with cell wall skeletons of
Mycobacterium Bovis-Bacillus Calmette-Guerin: Experimental and clinical results.
Ann. NY Acad. Sci., 277, 209-227.
2) Azuma, I, et al. (1974): Biologically active components from mycobaterial cell walls. I.
Isolation and composition of cell wall skeleton and component P3. J. Nat. Cancer Inst.,
52, 94-101.
3) Murray, H. W. et al. (1984): Impaired production of lymphokines and immune (gamma)
interferon in the acquired immunodeficiency syndrome. N. Engl. J. Med., 310, 883-889.
4) Basham, T., and Merigan, T. C. (1982): Immunoregulation by gamma-interferon?
Nature, 299, 778.
5) Nastala, C. L, et al. (1994): Recombinant IL-12 administration induces tumor regressionin association with IFN-y production. J. Immunol., 153, 1697-1706.
6) Yamamura, Y. et al. (1983): Randomized controlled study of adjuvant immunotherapy
with Nocardia rubra cell wall skeleton for inoperable lung cancer. Cancer Res., 43,5573-5579.