Intralymphatic Iniection of BeG Into Rhesus Monkeys
1
Charles E. Mangan, M.D.,2 K. Ann Jeglum, B.S.,a Thomas V. Sedlacek, M.D.,4 James E. Wheeler, M.D.,5 and John J. Mikuta, M.D.;;' 7 SUMMARY-Since the route of administration of BCG may have an important function in immunotherapy, we investigated intra lymphatic administration to direct BCG to the lymph nodes. Multiple injections of high doses of BCG were administered to 6 rhesus monkeys via the dorsal lymphatics of the lower limb. A suppurative lymphadenitis was observed along the lower limb and in the inguinal area in 5 of the 6 monkeys. However, many of the complications reported with other routes of administration were not observed. Granulomatous reactions and histiocytic responses developed in lymph nodes on the injected sides of the pelvis and distant nodes as well as in the liver. The intralymphatic route is the method by which high doses of nonspecific immunostimulants were delivered to regional lymph nodes. The efficacy of this approach remains to be established In tumor-bearing animals and humans.-J Natl Cancer Inst 55: 699-704, 1975.
The results of immunotherapy may depend on the route by which immunostimulants are administered. In experimental systems, BCG may cause local regression of tumors and, in some instances, curtail metastasis in distant sites or in regional lymph nodes (1,2). Treatment of experimental animal models and humans include sc and intralesional (3, 4) iv (5-7). ip (8-9), oral (10), scarification (11, 12), aerosol (13). and. recently, intranodal routes of administration (Bast RC, Zbar B. Smith H, et al: Personal communication). Mycobacteria have caused sinus hyperplasia. histiocytosis, and granulomatosis in lymph nodes (14-17). At present, it is unclear how immunostimulants such as BCG reach the regional or distal lymph nodes when administered by these routes. Lymphocytes may carry the organisms from the injection site to the draining lymph node. Alternatively, lymphocytes may be sensitized at the site of BCG injection and then release mediators of cellular immunity (2, 18). An important concept in favor of the intralymphatic route of administration derives from one of the basic factors in immunotherapy of cancer: the advantage of contact between tumor cells and the immunostimulating agent. Clinical utilization of intralesional injection of BCG in malignant melanoma emphasized the efficacy of this principle (4, 17, 19, 20). An inflammatory reaction had a significant mechanism in the regression of localized lesions (17). Furthermore, Hanna et al. (15) and Snodgrass and Hanna (16) emphasized the importance of granulomatous reactions in elimination of tumor cells and prevention of metastasis in regional lymph nodes. Therefore, could the efficacy of systemic immunotherapy be improved by the transport of BCG directly to the lymph node? The present report concerns this possibility and the intralymphatic administration of BCG with particular emphasis on the reaction in lymph nodes. Many experimental systems may not approximate the patient in the clinic because of anatomic differences and physiologic variations in normal animals' immune response. We have chosen to investigate the hypothesis in the rhesus monkey because of its close phylogeny to man. It is essential to determine whether this direct mode of
administration causes any severe adverse effects in a primate before its value in destroying tumor cells both in animal models and human patients can be evaluated. MATERIALS AND METHODS
Animals and testing.-Adult female rhesus monkeys (7-12 yr) were given an antigen panel including histoplasmin, streptococcus, purified protein derivative, coccidiosis, and mumps to exclude active tuberculosis and to demonstrate immunocompetence. At this time, preliminary blood studies including complete blood count, d~f ferential, and biochemical studies [albumin: globuhn ratio, total protein, albumin, globulin, total bilirubin, calcium, total cholesterol. glucose inorganic phosphorus, serum glutamic-oxaloacetic transaminase, urea nitrogen, uric acid, alkaline phosphatase, and lactate dehydrogenase (LDH)] were performed. BCG.-The Glaxo-Lilly strain of BCG (Eli Lilly & Co., Indianapolis. Ind.) containing 4-9 million o~ganism~/.ml sterile water was used. The normal human Immumzmg dose, as reported by the manufacturer, is 0.1 ml by intradermal (id) injection. Method of administration.-All monkeys were sensitized with 0.1 ml BCG by id injection into the forelimbs. We had three schedules of inoculation: once a week, once every 2 weeks, and once every 3 weeks. The appearance of an erythematous reaction determined that t~e inoculations given every 14 days yielded the most rapid sensitization. By the 28th day, all animals exhibited erythema and induration followed by mild ulceration at the injection site. After the induction of the delayed hypersensitivity reaction. 6 monkeys were divided into 3 experimental groups involving dose variables and the number of intralymphatic injections to be administered. The two monkeys in each group were administered 20, 50, or 100 times the normal immunizing dose on schedules of one, two, or three intralymphatic injections (text-fig. 1). Before the intralymphatic cannulation, we drew blood and recorded the rectal temperatures. Then each monkey was anesthetized with 10 mg Sernylan (phencyclidine hydrochloride; Bio-Ceutic Laboratories, Inc., St. Joseph, Mo.), and 3 ml Alphaazurine 2G dye (Allied Chemical Corp., Morristown, N ..J.) was injected into the webs of Received March 20. 1975; accepted May 13, 1975. Assistant Professor of Obstetrics and Gynecology, University of Pennsylvania School of Medicine. Address reprint requests to Charles E. Mangan, M.D., 3400 Spruce St., Philadelphia, Pa. 19104. 3 University of Pennsylvania School of Veterinary Medicine. 4 Instructor, Department of Obstetrics and Gynecology, and Junior Clinical Fellow, Gynecologic Oncology, Hospital of the University of Pennsylvania. 5 Assistant Professor, Department of Surgical Pathology. Hospital of the University of Pennsylvania. 6 Director of Gynecologic Oncology, Professor of Obstetrics and Gynecology, University of Pennsylvania School of Medicine. 1 The authors thank Dr. Luigi Mastroianni for his permission to use the facilities of the Division of Reproductive Biology; Dr. Arnold Chait for his assistance in lymphangiography; Mr. Charles Meltzer, Mr. Fred Norman, and Mrs. Joan Trammell for their technical assistance; and Miss Barbara Erwins for preparation of the manuscript. 1 2
JOURNAL OF THE NATIONAL CANCER INSTITUTE, VOL. 55, NO.3, SEPTEMBER 1975
699
700
MANGAN ET AL.
MONKEY # .. :: intradermal administration d
•
• 50X
2
o I Gloxo-Lilly BeG {"""",I ;"""';ring dose>
.:: In1rolymphotic injection
...
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•
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~::::j=;i:::=::::;;1;#:::=======::::::;*-pPos, mortem lOX 10 X right side
80
'ett side
100 120 DAYS
140
160
180
200
220
TEXT·FIGURE I.-Time schedule and dosages of intralymphatic injections of BeG given to 6 monkeys.
the first two digits of the lower extremities. We used the standard lymphangiography techniques as described for humans (21) and for dogs (22). A single dorsal lymphatic vessel along the tibia was cannulated with a 30-gauge needle, and the selected dose of BeG (diluted to 4 ml with sterile water) was injected. Animals were examined daily. Three of the 6 monkeys received rechallenge injections of 10 times the immunizing dose at a time when ulcerations had healed and the lymphatics were suitable for recannulation.
RESULTS Clinical
Upon completion of intralymphatic administration of BeG into 6 monkeys, 5 survived without pathologic effects, but 1 died 10 days after the first intralymphatic injection. The mean rectal temperatures for all monkeys (text-fig. 2) ranged from 101.0° to 102.8° F (normal range is 102.5° to 103.5° F) (23). Blood studies were performed on each monkey once a week for 3 weeks before the initial intralymphatic admin-
98°~~-+--~+-4-~-4--~+-4-~-+~~
o
2 4 6 8 10 12 14 16 18 20 22 24 26 28
DAYS TEXT-FIGURE 2.-Mean values of rectal temperatures for 6 monkeys taken twice daily before intralymphatic injections and for 28 subsequent days.
istration and once a week subsequently (tables lA, B) . The normal blood values of rhesus monkeys vary greatly [as discussed by HilI (24) and as exemplified by our data prior to treatment]. Alkaline phosphatase and LDH in 5 of the 6 animals were increased. These elevated values indicated some degree of tissue damage and inflammatory process withi~ the liver, as indicated by granu· lomas which may be expected from the administration of BeG. Although the total white blood cell count appeared to stay within a normal range in the differential, there was a slight increase in segmented neutrophils and a decrease in lymphocytes. It is difficult to attach any significance to these variations, considering the wide range of values recorded before and after the BeG was injected into the lymphatics. Four of the 5 monkeys had localized ulceration of 1-4 cm along the lymphatic vessel that was used for cannulation and in the inguinal area. Monkey #2, which showed abscesses all along the injected lymphatic vessel, had undergone a more extensive regimen involving 4 consecutive weekly O.l-ml inoculations followed by 3 id immunizations every 14 days. At this time, no conclusion can be drawn concerning the significance of the sensitization schedule and its effects upon the anamnestic response. Histopathologic
Monkey #1 was given an injection on one side with 50 times the normal human immunizing dose and died 10 days later. The cause of death could not be determined from histologic studies. Larvae of Pneumonyssus simicola and the associated characteristic pigment were in the lungs. Rare granulomas were present in an otherwise normal-appearing liver. Hypertrophy of the inguinal lymph nodes with a marked histiocytic response and early granuloma formation were observed on the injected but not on the contralateral side. The kidneys were unremarkable. Monkey #5 was killed 125 days after the last intralymphatic injection for histopathologic examination. This animal had received 2 injections on the right side, and the right inguinal and pelvic nodes had central necrosis rimmed by epithelioid cells and fibroblasts. Sinus tracts connected the right inguinal nodes with crusted ulcerations in the overlying skin. The lungs contained mites consistent with Pneumonyssus simicola. No granulomas were found on the contralateral side or in multiple nodes above the pelvis. Monkey #6, which initially had received bilateral intralymphatic injection and then was rechallenged on the right side and later on the left side, was killed 2 weeks after the third intralymphatic injection of BeG. Suppurative skin ulcerations were present in the left inguinal and popliteal areas; enlarged left inguinal, left pelvic, and periaortic lymph nodes contained focally necrotic granulomas. Other lymph nodes were unremarkable except for rare epithelioid granulomas in the right pelvic and left axillary nodes. The viscera of monkeys #5 and 6 were microscopically normal except for the liver, which contained numerous noncaseating granulomas composed of epithelioid cells and lymphocytes. Acid-fast stains of involved lymph nodes and liver were negative in monkeys #5 and 6, but auramine-rhodamine staining demonstrated organisms consistent with BeG in the right inguinal node of monkey #5 and in the subcutaneous tissue in the ulcerated area overlying the left
%_
",3 ______________ _
5.177XI0' (4.8X 10'-5.49 X 10') 11.1 (10A-ll.7) 36.1 (34.4-37.1) 69 (66-76) 21.8 (20.1-24.2) 31.2 (30.2-32.3) 163 (152-175) 0.2 (0.1-0.3) 68 (40-116) 7.2 (6.9-7.9) 1.0 (0.8-1.1) 3.7 (3.6-3.8) 3.5 (3.3-4.2)
3 (3-4) 0
0
41
56
4,875 (3,500-6,100) 0
Beforea
Monkey #1
3.8
3.8
1.0
7.6
53
0.3
190
33.0
22.2
69
31.5
9.8
4.55XlO&
0
0
2
17
81
0
5,400
Aftera
59 (57-62) 7.6 (7.2-7.8) 1.2 (1.1-1.4) 4.5 (4.1-5.0) 3.1 (2.8-3.3) 10.1 (10.0-10.8) 155 (140-175) 622 (575-670) 4.7 (2.6-7.0) 19 (19-20) 0.3 (0.2-0.3)
4.98XI0' (4.8 X 10 6-5.3 X lOG) 11.6 (11.3-12.2) 36.5 (35.7-38.1) 73 (72-74) 23.9 (23.2-24.0) 32.0 (31.6-32.5) 151 (110-180) 0.2
7,866 (7,800-8,000) 1 (0-2) 47.3 (34-57) 44 (30-58) 2 (1-3) 6 (3-12) 0
Beforea Afterb
(42-70) 8.3 (7.5-9.1) 1.0 (0.8-1.2) 4.1 (3.9-4.3) 4.2 (3.5-4.8) 9.0 (6.2-10.3) 159 (156-175) 1,126 (870-1,550) 5.5 (4.3-6.9) 17 (13-22) 0.5 (0.2-0.9)
54
5.44 X 106 (4.88 X 106-6.05 X 10') 11.9 (10.5-12.8) 38.8 (34.5-43.8) 71 (67-81) 22.2 (20.7-24.0) 31.2 (29.0-32.8) 315 (150-483) 0.2
4.2 (1-8) 0
(0-6)
52.4 (38-66) 40.2 (26-56) 3
10,660 (7,200-14,600) 0
Monkey #2 Beforea
67 (60-75) 8.8 (8.3-9.6) 0.9 (0.8-1.0) 4.3 (4.1-4.5) 4.5 (4.2-5.3) 10.8 (10.5-11.0) 165 (150-175) 900 (750-1,000) 6.5 (5.7-7.0) 17 (16-18) 0.2 (0.2-0.3)
5.236XI0G (4. 76XIO'-5 .54XI0') 11.6 (11.1-11.9) 37.6 (37.3-38.0) 71 (67-79) 22.2 (21.3-23.4) 30.9 (29.9-31.7) 135 (120-173) 0.2
4 (0-8) 0
50 (43-55) 44 (34-57) 0
Afterb
65 (60-70) 8.9 (8.7-9.1) 0.7 (0.7-0.8) 4.0 (3.8-4.1) 5.0 (4.6-5.3) 10.3 (10.2-10.5) 170 (160-180) 1,200 (1,150-1,250) 5.9 (5.5-6.2) 20 (17-24) 0.4 (0.3-0.5)
5.15XIOG (4.92 X 106-5.38 X 106) 11.2 (10.7-11. 7) 34.9 (32.9-37.0) 68 (67-70) 21.6 (21.6-21.7) 31.7 (30.7-32.7) 215 (175-256) 0.2
9,250 (7 ,300-11 ,200) 1 (0-1) 50 (40-61, 41 (36-47) 0 (0-1) 7 (2-12) 0
Monkey #3
8,833 (7,500-10,700) 0
lA.-Mean blood values of monkeys #1,2, and 3 befure and after intralymphatic injections
• Mean value 3 weeks before intralymphatic injection. • Mean value after intralymphatic injection for 3 week., then monthly.
Uric acid, mg/l00 mL ___________________ _
Urea nitrogen, mg/l00 mI ___ _
Inorganic phosphorus, mg/loo mI __________ _
Lactate dehydrogenase, nm/mI ____________ _
Total cholesterol, mg/loo mL _____________ _
Calcium, mg/loo mL ____________________ _
Globulin, g/loo mI ___________ _
Albumin, g/Ioo mI _________ _
Albumin:globulin ratio _____ _
Serum glutamic-oxaloacetic transaminase, nm/ml. Total protein, g/loo mL _________________ _
Total bilirubin, mg/IOO mL __ _
Alkaline phosphatase, nm/mL ____________ _
%.
Mean corpuscular hemoglobin concentration,
Mean corpuscular hemoglobin, pg __________ _
Mean corpuscular volume,
Hematocrit,
Hemoglobin, g/IOO mL ___________________ _
Red blood cells, mm' _____________________ _
%__________________________ _
%_____ _
Eosinophils,
Basophils,
%________ _
%__
Monocytes,
Lymphocytes, % __ _
Segmented neutrophils,
Band neutrophils, %___________________ _
White blood cells, mml_
Blood determinations
TABLE
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t'I
~
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Nl
INJECTION OF BCG INTO RHESUS MONKEYS
inguinal nodes of monkey #6. Mycobacterial cultures of involved lymph noaes of monkeys #5 and 6 were negative except for rare colonies (grown from one node of #6) that had the biochemical characteristics of Mycobacterium bovis. DISCUSSION
In a determination of the validity of a new administration route of a nonspecific immunostimulant (i.e., intralymphatic), the initial consideration must be the safety of the patient. In these studies, no adverse clinical effects were found after intralymphatic injection of BCG in 5 of the 6 monkeys. The autopsy results of monkey #1 showed no apparent cause of death. It may be significant that before the intralymphatic injection, this animal appeared emaciated and lethargic in comparison to the other monkeys. The clinical course of this animal was possibly affected by BCG, i.e., by influencing the lung mite infestation. However, higher and multiple intralymphatic injections in the subsequent 5 monkeys had no adverse effect. Reviews of the current literature indicate that intralymphatic administration should present no greater risks than other routes. In these monkeys, we did not observe the systemic complications such as fever, malaise, and influenza-like syndrome reported in previous studies on humans. Blood and temperature values fluctuated within a normal range. Although possibly related to hepatic granulomas, the elevated alkaline phosphatase and LDH values were not associated with any obvious pathology. Elevated alkaline phosphatase values were previously reported with BCG immunotherapy (13). Unfortunately, the normal range of values in rhesus monkeys for LDH and alkaline phosphatase have not been established, but before the experiment, they were higher than normal human values. Complications varying from localized abscess to severe hypersensitivity including anaphylaxis have been reported with the intralesional administration of BCG (25). We observed only ulcerations along the lymphatic vessel used for injection and in the inguinal area. Regional lymphadenitis with suppuration was a complication of BCG vaccination for tuberculosis (26). Nevertheless, monkey #4 received 100 times the normal human immunizing dose and rechallenge and showed no ulceration even 6 months later. Several major factors must be considered in any type of immunotherapy. The dose of immunostimulants and level of reticuloendothelial system activity are important. Results from other investigations consistently indicate that higher doses yield greater percentages of regression of neoplasms in human and animal studies (18, 27). The route of administration may also influence the outcome of immunopotentiation as evidenced by scarification, which results in a higher number of viable organisms (12, 28). Localization or dissemination of the BCG may also be important. In both experimental animals and clinical trials, whether distant metastasis can be eliminated by the administration of an immunostimulant has been questioned. Intralymphatic administration may allow for systemic distribution of higher doses of BCG to many lymph nodes. The histiocytic response and granulomatous reaction are significant in the host's immunologic defense against malignant cells (14, 17). The BCG-mediated granulomatous reaction may be enhanced by administration that
703
results in intimate contact between BCG, tumor, and lymph node cells. Indeed, these studies demonstrated that the intralymphatic injection of BCG could induce profound granulomatosis in lymph nodes along the injected lymphatic vessel, in distal nodes, and in the liver. Intralymphatic injection of immunostimulants may offer another route of administration for systemic immunopotentiators. The immediate transport to the lymph node and the systemic dissemination of a higher number of organisms is deemed advantageous. REFERENCES (1) BAST Re, ZBAR B, BoRSos T, et al: BCG and cancer. Part I. N Engl J Med 290: 1413-1420, 1974 (2) ZBAR B, BERNSTEIN ID, BARTLETT GL, et al: Immunotherapy of cancer: Regression of intradennal tumors and prevention of growth of lymph node metastasis after intralesional injection of living Mycobacterium bovis. J Natl Cancer Inst 49: 119-130, 1972
(3) ZBAR B, TANAKA T: Immunotherapy of cancer: Regression of tumors after intralesional injection of Jiving Mycobacterium bovis. Science 172:271-273, 1971 (4) MORTON DL, EILBER FR, MALMGREN RA, et al: Immunological factors which influence response to immunotherapy in malignant melanoma. Surgery 68:154-164, 1970 (5) BALDWIN RW, DIMM MV: BCG immunotherapy of pulmonary growths from intravenously transferred rat tumour cells. Br J Cancer 27:48-54, 1973 (6) HOUCHENS DP, GOLDBERG AL, GASTON MR, et al: Studies of the effects of Bacillus Calmette-Guerin on Moloney sarcoma virus-induced tumors in normal and immunosuppressed mice. Cancer Res 33:685-690, 1973 (7) LARSON CL, USHIGIMA RN, FLOREY MJ, et al: Effects of Friend disease virus in mice. Nature [New Bioi] 229:243244, 1971 (8) LAPPE MA, PREHN RT: Immunologic surveillance at the mac-
(9)
(10)
(11)
(12)
(H)
(14) (15)
(16)
(17)
(18)
(19) (20)
roscopic level: Nonselective elimination of premalignant skin papillomas. Cancer Res 29:2374-2378, 1969 FALK RE: Use of oral and intraperitoneal BCG in the treatment of metastatic melanoma and adenocarcinoma. In Neoplasm and Immunity: Theory and Application. Proceedings of the Second Annual Chicago Symposium, Evanston, Ill., Schori Press, 1974 FALK RE, MANN P, LANGER B: Cell-mediated immunity to human tumors. Abrogation by serum factors and nonspecific effects of oral BCG therapy. Arch Surg 107:261-265, 1973 DEVRIES JE, RUMKE P: Immunological studies in melanoma patients treated with BCG. Br J Cancer 28:97-102, 1973 GUTTERMAN JU, MAVLlGIT GM, McBRIDE C, et al: Active immunotherapy with BCG for recurrent malignant melanoma. Lancet 1:1208--1212, 1973 LIPTON A, GARNER FB, MEYER CA: Aerosol BCG treatment of pulmonary metastasis-a phase I study. In Neoplasm Immunity: BCG Vaccination. Proceedings of the First Annual Chicago Symposium, Evanston, Ill., Schori Press, 1973, pp 79-85 BLACK MN, SPEER FD: Sinus histiocytosis of lymph nodes in cancer. Surg Gynecol Obstet 106: 163--175, 1958 HANNA MG, ZBAR B, RAPP HJ. et al: Histopathology of tumor regression after intralesional injection of Mycobacterium bovis. I. Tumor growth and metastasis. J Natl Cancer Inst 48:1441-1455, 1972 SNODGRASS MJ, HANNA MG: Ultrastructural studies of histiocyte-tumor cell interactions during tumor regression after intralesional injection of Mycobacterium bovis. Cancer Res 33:701-716, 1973 MASTRANGELO MJ, KIM YH, BORNSTEIN RS, et al: Clinical and histiologic correlation of melanoma regression after intralesional BCG therapy: A case report. J Natl Cancer Inst 52:19-24, 1974 GUTTERMA:-I JU, MAVLlGIT GB, McBRIDE C, et al: Immuno· prophylaxis of malignant melanoma with systemic BCG: Study of strain, dose, and schedule. Natl Cancer Inst Monogr 39:205-212, 1973 PINSKY CM, HIRSHAUT Y, OETTGEN HF: Treatment of malignant melanoma by intratumoral injection of BCG. Nat! Cancer Inst Monogr 39:225-228, 1973 BORNSTEIN RS, MASTRANGELO MJ, SULlT H, et al: Immuno-
704
(21) (22) (23) (24)
MANGAN ET AL. therapy of melanoma with intralesional BCG. Natl Cancer Inst Monogr 39:213-220, 1973 KIN MOUTH JB, TAYLOR GW, HARPER RA: Lymphangiography technique for its clinical use in lower limbs. Br Med J 1:940-942, 1955 PRIER JE, ScHAFFER B, SKELLEY JE: Direct lymphangiography in the dog. J Am Vet Med Assoc 140:943-947, 1962 GISLER DB, BENSON RE, YOUNG RJ, et al: Colony husbandry of research monkeys. Ann NY Acad Sci 85:758-768, 1960 HILL WC: Primates: Comparative Anatomy and Taxonomy. VI. Cercopithecoidea. Edinburgh, Edinburgh Univ Press, 1966, p 84
(25) BAST RC, ZBAR B, BORSOS T, et al: BCG and cancer. Part 2.
N Engl J Med 290:1458-1469, 1974 (26) SPARKS FC, SILVERSTEIN MJ, HUNT JS, et al: Complications
of BCG immunotherapy in patients with cancer. N Engl J Med 289:827-830, 1973 (27) BLUMING AZ, VOGEL CL, ZIEGLER JL, et aI: Immunological effects of BCG in malignant melanoma: Two modes of administration compared. Ann Intern Med 76:405-411, 1972 (28) KLEIN E, HOLTERMANN OA, P APERMASTER B, et al: Immunologic approaches to various types of cancer with the use of BCG and purified protein derivatives. Nat! Cancer lnst Monogr 39:229-242, 1973
1
Charles E. Mangan, M.D.,2 K. Ann Jeglum, B.S.,a Thomas V. Sedlacek, M.D.,4 James E. Wheeler, M.D.,5 and John J. Mikuta, M.D.;;' 7 SUMMARY-Since the route of administration of BCG may have an important function in immunotherapy, we investigated intra lymphatic administration to direct BCG to the lymph nodes. Multiple injections of high doses of BCG were administered to 6 rhesus monkeys via the dorsal lymphatics of the lower limb. A suppurative lymphadenitis was observed along the lower limb and in the inguinal area in 5 of the 6 monkeys. However, many of the complications reported with other routes of administration were not observed. Granulomatous reactions and histiocytic responses developed in lymph nodes on the injected sides of the pelvis and distant nodes as well as in the liver. The intralymphatic route is the method by which high doses of nonspecific immunostimulants were delivered to regional lymph nodes. The efficacy of this approach remains to be established In tumor-bearing animals and humans.-J Natl Cancer Inst 55: 699-704, 1975.
The results of immunotherapy may depend on the route by which immunostimulants are administered. In experimental systems, BCG may cause local regression of tumors and, in some instances, curtail metastasis in distant sites or in regional lymph nodes (1,2). Treatment of experimental animal models and humans include sc and intralesional (3, 4) iv (5-7). ip (8-9), oral (10), scarification (11, 12), aerosol (13). and. recently, intranodal routes of administration (Bast RC, Zbar B. Smith H, et al: Personal communication). Mycobacteria have caused sinus hyperplasia. histiocytosis, and granulomatosis in lymph nodes (14-17). At present, it is unclear how immunostimulants such as BCG reach the regional or distal lymph nodes when administered by these routes. Lymphocytes may carry the organisms from the injection site to the draining lymph node. Alternatively, lymphocytes may be sensitized at the site of BCG injection and then release mediators of cellular immunity (2, 18). An important concept in favor of the intralymphatic route of administration derives from one of the basic factors in immunotherapy of cancer: the advantage of contact between tumor cells and the immunostimulating agent. Clinical utilization of intralesional injection of BCG in malignant melanoma emphasized the efficacy of this principle (4, 17, 19, 20). An inflammatory reaction had a significant mechanism in the regression of localized lesions (17). Furthermore, Hanna et al. (15) and Snodgrass and Hanna (16) emphasized the importance of granulomatous reactions in elimination of tumor cells and prevention of metastasis in regional lymph nodes. Therefore, could the efficacy of systemic immunotherapy be improved by the transport of BCG directly to the lymph node? The present report concerns this possibility and the intralymphatic administration of BCG with particular emphasis on the reaction in lymph nodes. Many experimental systems may not approximate the patient in the clinic because of anatomic differences and physiologic variations in normal animals' immune response. We have chosen to investigate the hypothesis in the rhesus monkey because of its close phylogeny to man. It is essential to determine whether this direct mode of
administration causes any severe adverse effects in a primate before its value in destroying tumor cells both in animal models and human patients can be evaluated. MATERIALS AND METHODS
Animals and testing.-Adult female rhesus monkeys (7-12 yr) were given an antigen panel including histoplasmin, streptococcus, purified protein derivative, coccidiosis, and mumps to exclude active tuberculosis and to demonstrate immunocompetence. At this time, preliminary blood studies including complete blood count, d~f ferential, and biochemical studies [albumin: globuhn ratio, total protein, albumin, globulin, total bilirubin, calcium, total cholesterol. glucose inorganic phosphorus, serum glutamic-oxaloacetic transaminase, urea nitrogen, uric acid, alkaline phosphatase, and lactate dehydrogenase (LDH)] were performed. BCG.-The Glaxo-Lilly strain of BCG (Eli Lilly & Co., Indianapolis. Ind.) containing 4-9 million o~ganism~/.ml sterile water was used. The normal human Immumzmg dose, as reported by the manufacturer, is 0.1 ml by intradermal (id) injection. Method of administration.-All monkeys were sensitized with 0.1 ml BCG by id injection into the forelimbs. We had three schedules of inoculation: once a week, once every 2 weeks, and once every 3 weeks. The appearance of an erythematous reaction determined that t~e inoculations given every 14 days yielded the most rapid sensitization. By the 28th day, all animals exhibited erythema and induration followed by mild ulceration at the injection site. After the induction of the delayed hypersensitivity reaction. 6 monkeys were divided into 3 experimental groups involving dose variables and the number of intralymphatic injections to be administered. The two monkeys in each group were administered 20, 50, or 100 times the normal immunizing dose on schedules of one, two, or three intralymphatic injections (text-fig. 1). Before the intralymphatic cannulation, we drew blood and recorded the rectal temperatures. Then each monkey was anesthetized with 10 mg Sernylan (phencyclidine hydrochloride; Bio-Ceutic Laboratories, Inc., St. Joseph, Mo.), and 3 ml Alphaazurine 2G dye (Allied Chemical Corp., Morristown, N ..J.) was injected into the webs of Received March 20. 1975; accepted May 13, 1975. Assistant Professor of Obstetrics and Gynecology, University of Pennsylvania School of Medicine. Address reprint requests to Charles E. Mangan, M.D., 3400 Spruce St., Philadelphia, Pa. 19104. 3 University of Pennsylvania School of Veterinary Medicine. 4 Instructor, Department of Obstetrics and Gynecology, and Junior Clinical Fellow, Gynecologic Oncology, Hospital of the University of Pennsylvania. 5 Assistant Professor, Department of Surgical Pathology. Hospital of the University of Pennsylvania. 6 Director of Gynecologic Oncology, Professor of Obstetrics and Gynecology, University of Pennsylvania School of Medicine. 1 The authors thank Dr. Luigi Mastroianni for his permission to use the facilities of the Division of Reproductive Biology; Dr. Arnold Chait for his assistance in lymphangiography; Mr. Charles Meltzer, Mr. Fred Norman, and Mrs. Joan Trammell for their technical assistance; and Miss Barbara Erwins for preparation of the manuscript. 1 2
JOURNAL OF THE NATIONAL CANCER INSTITUTE, VOL. 55, NO.3, SEPTEMBER 1975
699
700
MANGAN ET AL.
MONKEY # .. :: intradermal administration d
•
• 50X
2
o I Gloxo-Lilly BeG {"""",I ;"""';ring dose>
.:: In1rolymphotic injection
...
#::Rechollenge introlymphotic injection Livir-J
rj:g~jde flflffg
•
living
J::~;t=;1:=========:::]POSf mort~
~::::j=;i:::=::::;;1;#:::=======::::::;*-pPos, mortem lOX 10 X right side
80
'ett side
100 120 DAYS
140
160
180
200
220
TEXT·FIGURE I.-Time schedule and dosages of intralymphatic injections of BeG given to 6 monkeys.
the first two digits of the lower extremities. We used the standard lymphangiography techniques as described for humans (21) and for dogs (22). A single dorsal lymphatic vessel along the tibia was cannulated with a 30-gauge needle, and the selected dose of BeG (diluted to 4 ml with sterile water) was injected. Animals were examined daily. Three of the 6 monkeys received rechallenge injections of 10 times the immunizing dose at a time when ulcerations had healed and the lymphatics were suitable for recannulation.
RESULTS Clinical
Upon completion of intralymphatic administration of BeG into 6 monkeys, 5 survived without pathologic effects, but 1 died 10 days after the first intralymphatic injection. The mean rectal temperatures for all monkeys (text-fig. 2) ranged from 101.0° to 102.8° F (normal range is 102.5° to 103.5° F) (23). Blood studies were performed on each monkey once a week for 3 weeks before the initial intralymphatic admin-
98°~~-+--~+-4-~-4--~+-4-~-+~~
o
2 4 6 8 10 12 14 16 18 20 22 24 26 28
DAYS TEXT-FIGURE 2.-Mean values of rectal temperatures for 6 monkeys taken twice daily before intralymphatic injections and for 28 subsequent days.
istration and once a week subsequently (tables lA, B) . The normal blood values of rhesus monkeys vary greatly [as discussed by HilI (24) and as exemplified by our data prior to treatment]. Alkaline phosphatase and LDH in 5 of the 6 animals were increased. These elevated values indicated some degree of tissue damage and inflammatory process withi~ the liver, as indicated by granu· lomas which may be expected from the administration of BeG. Although the total white blood cell count appeared to stay within a normal range in the differential, there was a slight increase in segmented neutrophils and a decrease in lymphocytes. It is difficult to attach any significance to these variations, considering the wide range of values recorded before and after the BeG was injected into the lymphatics. Four of the 5 monkeys had localized ulceration of 1-4 cm along the lymphatic vessel that was used for cannulation and in the inguinal area. Monkey #2, which showed abscesses all along the injected lymphatic vessel, had undergone a more extensive regimen involving 4 consecutive weekly O.l-ml inoculations followed by 3 id immunizations every 14 days. At this time, no conclusion can be drawn concerning the significance of the sensitization schedule and its effects upon the anamnestic response. Histopathologic
Monkey #1 was given an injection on one side with 50 times the normal human immunizing dose and died 10 days later. The cause of death could not be determined from histologic studies. Larvae of Pneumonyssus simicola and the associated characteristic pigment were in the lungs. Rare granulomas were present in an otherwise normal-appearing liver. Hypertrophy of the inguinal lymph nodes with a marked histiocytic response and early granuloma formation were observed on the injected but not on the contralateral side. The kidneys were unremarkable. Monkey #5 was killed 125 days after the last intralymphatic injection for histopathologic examination. This animal had received 2 injections on the right side, and the right inguinal and pelvic nodes had central necrosis rimmed by epithelioid cells and fibroblasts. Sinus tracts connected the right inguinal nodes with crusted ulcerations in the overlying skin. The lungs contained mites consistent with Pneumonyssus simicola. No granulomas were found on the contralateral side or in multiple nodes above the pelvis. Monkey #6, which initially had received bilateral intralymphatic injection and then was rechallenged on the right side and later on the left side, was killed 2 weeks after the third intralymphatic injection of BeG. Suppurative skin ulcerations were present in the left inguinal and popliteal areas; enlarged left inguinal, left pelvic, and periaortic lymph nodes contained focally necrotic granulomas. Other lymph nodes were unremarkable except for rare epithelioid granulomas in the right pelvic and left axillary nodes. The viscera of monkeys #5 and 6 were microscopically normal except for the liver, which contained numerous noncaseating granulomas composed of epithelioid cells and lymphocytes. Acid-fast stains of involved lymph nodes and liver were negative in monkeys #5 and 6, but auramine-rhodamine staining demonstrated organisms consistent with BeG in the right inguinal node of monkey #5 and in the subcutaneous tissue in the ulcerated area overlying the left
%_
",3 ______________ _
5.177XI0' (4.8X 10'-5.49 X 10') 11.1 (10A-ll.7) 36.1 (34.4-37.1) 69 (66-76) 21.8 (20.1-24.2) 31.2 (30.2-32.3) 163 (152-175) 0.2 (0.1-0.3) 68 (40-116) 7.2 (6.9-7.9) 1.0 (0.8-1.1) 3.7 (3.6-3.8) 3.5 (3.3-4.2)
3 (3-4) 0
0
41
56
4,875 (3,500-6,100) 0
Beforea
Monkey #1
3.8
3.8
1.0
7.6
53
0.3
190
33.0
22.2
69
31.5
9.8
4.55XlO&
0
0
2
17
81
0
5,400
Aftera
59 (57-62) 7.6 (7.2-7.8) 1.2 (1.1-1.4) 4.5 (4.1-5.0) 3.1 (2.8-3.3) 10.1 (10.0-10.8) 155 (140-175) 622 (575-670) 4.7 (2.6-7.0) 19 (19-20) 0.3 (0.2-0.3)
4.98XI0' (4.8 X 10 6-5.3 X lOG) 11.6 (11.3-12.2) 36.5 (35.7-38.1) 73 (72-74) 23.9 (23.2-24.0) 32.0 (31.6-32.5) 151 (110-180) 0.2
7,866 (7,800-8,000) 1 (0-2) 47.3 (34-57) 44 (30-58) 2 (1-3) 6 (3-12) 0
Beforea Afterb
(42-70) 8.3 (7.5-9.1) 1.0 (0.8-1.2) 4.1 (3.9-4.3) 4.2 (3.5-4.8) 9.0 (6.2-10.3) 159 (156-175) 1,126 (870-1,550) 5.5 (4.3-6.9) 17 (13-22) 0.5 (0.2-0.9)
54
5.44 X 106 (4.88 X 106-6.05 X 10') 11.9 (10.5-12.8) 38.8 (34.5-43.8) 71 (67-81) 22.2 (20.7-24.0) 31.2 (29.0-32.8) 315 (150-483) 0.2
4.2 (1-8) 0
(0-6)
52.4 (38-66) 40.2 (26-56) 3
10,660 (7,200-14,600) 0
Monkey #2 Beforea
67 (60-75) 8.8 (8.3-9.6) 0.9 (0.8-1.0) 4.3 (4.1-4.5) 4.5 (4.2-5.3) 10.8 (10.5-11.0) 165 (150-175) 900 (750-1,000) 6.5 (5.7-7.0) 17 (16-18) 0.2 (0.2-0.3)
5.236XI0G (4. 76XIO'-5 .54XI0') 11.6 (11.1-11.9) 37.6 (37.3-38.0) 71 (67-79) 22.2 (21.3-23.4) 30.9 (29.9-31.7) 135 (120-173) 0.2
4 (0-8) 0
50 (43-55) 44 (34-57) 0
Afterb
65 (60-70) 8.9 (8.7-9.1) 0.7 (0.7-0.8) 4.0 (3.8-4.1) 5.0 (4.6-5.3) 10.3 (10.2-10.5) 170 (160-180) 1,200 (1,150-1,250) 5.9 (5.5-6.2) 20 (17-24) 0.4 (0.3-0.5)
5.15XIOG (4.92 X 106-5.38 X 106) 11.2 (10.7-11. 7) 34.9 (32.9-37.0) 68 (67-70) 21.6 (21.6-21.7) 31.7 (30.7-32.7) 215 (175-256) 0.2
9,250 (7 ,300-11 ,200) 1 (0-1) 50 (40-61, 41 (36-47) 0 (0-1) 7 (2-12) 0
Monkey #3
8,833 (7,500-10,700) 0
lA.-Mean blood values of monkeys #1,2, and 3 befure and after intralymphatic injections
• Mean value 3 weeks before intralymphatic injection. • Mean value after intralymphatic injection for 3 week., then monthly.
Uric acid, mg/l00 mL ___________________ _
Urea nitrogen, mg/l00 mI ___ _
Inorganic phosphorus, mg/loo mI __________ _
Lactate dehydrogenase, nm/mI ____________ _
Total cholesterol, mg/loo mL _____________ _
Calcium, mg/loo mL ____________________ _
Globulin, g/loo mI ___________ _
Albumin, g/Ioo mI _________ _
Albumin:globulin ratio _____ _
Serum glutamic-oxaloacetic transaminase, nm/ml. Total protein, g/loo mL _________________ _
Total bilirubin, mg/IOO mL __ _
Alkaline phosphatase, nm/mL ____________ _
%.
Mean corpuscular hemoglobin concentration,
Mean corpuscular hemoglobin, pg __________ _
Mean corpuscular volume,
Hematocrit,
Hemoglobin, g/IOO mL ___________________ _
Red blood cells, mm' _____________________ _
%__________________________ _
%_____ _
Eosinophils,
Basophils,
%________ _
%__
Monocytes,
Lymphocytes, % __ _
Segmented neutrophils,
Band neutrophils, %___________________ _
White blood cells, mml_
Blood determinations
TABLE
.....
--.l
>-l
t'I
~
>~ >-Z
Nl
INJECTION OF BCG INTO RHESUS MONKEYS
inguinal nodes of monkey #6. Mycobacterial cultures of involved lymph noaes of monkeys #5 and 6 were negative except for rare colonies (grown from one node of #6) that had the biochemical characteristics of Mycobacterium bovis. DISCUSSION
In a determination of the validity of a new administration route of a nonspecific immunostimulant (i.e., intralymphatic), the initial consideration must be the safety of the patient. In these studies, no adverse clinical effects were found after intralymphatic injection of BCG in 5 of the 6 monkeys. The autopsy results of monkey #1 showed no apparent cause of death. It may be significant that before the intralymphatic injection, this animal appeared emaciated and lethargic in comparison to the other monkeys. The clinical course of this animal was possibly affected by BCG, i.e., by influencing the lung mite infestation. However, higher and multiple intralymphatic injections in the subsequent 5 monkeys had no adverse effect. Reviews of the current literature indicate that intralymphatic administration should present no greater risks than other routes. In these monkeys, we did not observe the systemic complications such as fever, malaise, and influenza-like syndrome reported in previous studies on humans. Blood and temperature values fluctuated within a normal range. Although possibly related to hepatic granulomas, the elevated alkaline phosphatase and LDH values were not associated with any obvious pathology. Elevated alkaline phosphatase values were previously reported with BCG immunotherapy (13). Unfortunately, the normal range of values in rhesus monkeys for LDH and alkaline phosphatase have not been established, but before the experiment, they were higher than normal human values. Complications varying from localized abscess to severe hypersensitivity including anaphylaxis have been reported with the intralesional administration of BCG (25). We observed only ulcerations along the lymphatic vessel used for injection and in the inguinal area. Regional lymphadenitis with suppuration was a complication of BCG vaccination for tuberculosis (26). Nevertheless, monkey #4 received 100 times the normal human immunizing dose and rechallenge and showed no ulceration even 6 months later. Several major factors must be considered in any type of immunotherapy. The dose of immunostimulants and level of reticuloendothelial system activity are important. Results from other investigations consistently indicate that higher doses yield greater percentages of regression of neoplasms in human and animal studies (18, 27). The route of administration may also influence the outcome of immunopotentiation as evidenced by scarification, which results in a higher number of viable organisms (12, 28). Localization or dissemination of the BCG may also be important. In both experimental animals and clinical trials, whether distant metastasis can be eliminated by the administration of an immunostimulant has been questioned. Intralymphatic administration may allow for systemic distribution of higher doses of BCG to many lymph nodes. The histiocytic response and granulomatous reaction are significant in the host's immunologic defense against malignant cells (14, 17). The BCG-mediated granulomatous reaction may be enhanced by administration that
703
results in intimate contact between BCG, tumor, and lymph node cells. Indeed, these studies demonstrated that the intralymphatic injection of BCG could induce profound granulomatosis in lymph nodes along the injected lymphatic vessel, in distal nodes, and in the liver. Intralymphatic injection of immunostimulants may offer another route of administration for systemic immunopotentiators. The immediate transport to the lymph node and the systemic dissemination of a higher number of organisms is deemed advantageous. REFERENCES (1) BAST Re, ZBAR B, BoRSos T, et al: BCG and cancer. Part I. N Engl J Med 290: 1413-1420, 1974 (2) ZBAR B, BERNSTEIN ID, BARTLETT GL, et al: Immunotherapy of cancer: Regression of intradennal tumors and prevention of growth of lymph node metastasis after intralesional injection of living Mycobacterium bovis. J Natl Cancer Inst 49: 119-130, 1972
(3) ZBAR B, TANAKA T: Immunotherapy of cancer: Regression of tumors after intralesional injection of Jiving Mycobacterium bovis. Science 172:271-273, 1971 (4) MORTON DL, EILBER FR, MALMGREN RA, et al: Immunological factors which influence response to immunotherapy in malignant melanoma. Surgery 68:154-164, 1970 (5) BALDWIN RW, DIMM MV: BCG immunotherapy of pulmonary growths from intravenously transferred rat tumour cells. Br J Cancer 27:48-54, 1973 (6) HOUCHENS DP, GOLDBERG AL, GASTON MR, et al: Studies of the effects of Bacillus Calmette-Guerin on Moloney sarcoma virus-induced tumors in normal and immunosuppressed mice. Cancer Res 33:685-690, 1973 (7) LARSON CL, USHIGIMA RN, FLOREY MJ, et al: Effects of Friend disease virus in mice. Nature [New Bioi] 229:243244, 1971 (8) LAPPE MA, PREHN RT: Immunologic surveillance at the mac-
(9)
(10)
(11)
(12)
(H)
(14) (15)
(16)
(17)
(18)
(19) (20)
roscopic level: Nonselective elimination of premalignant skin papillomas. Cancer Res 29:2374-2378, 1969 FALK RE: Use of oral and intraperitoneal BCG in the treatment of metastatic melanoma and adenocarcinoma. In Neoplasm and Immunity: Theory and Application. Proceedings of the Second Annual Chicago Symposium, Evanston, Ill., Schori Press, 1974 FALK RE, MANN P, LANGER B: Cell-mediated immunity to human tumors. Abrogation by serum factors and nonspecific effects of oral BCG therapy. Arch Surg 107:261-265, 1973 DEVRIES JE, RUMKE P: Immunological studies in melanoma patients treated with BCG. Br J Cancer 28:97-102, 1973 GUTTERMAN JU, MAVLlGIT GM, McBRIDE C, et al: Active immunotherapy with BCG for recurrent malignant melanoma. Lancet 1:1208--1212, 1973 LIPTON A, GARNER FB, MEYER CA: Aerosol BCG treatment of pulmonary metastasis-a phase I study. In Neoplasm Immunity: BCG Vaccination. Proceedings of the First Annual Chicago Symposium, Evanston, Ill., Schori Press, 1973, pp 79-85 BLACK MN, SPEER FD: Sinus histiocytosis of lymph nodes in cancer. Surg Gynecol Obstet 106: 163--175, 1958 HANNA MG, ZBAR B, RAPP HJ. et al: Histopathology of tumor regression after intralesional injection of Mycobacterium bovis. I. Tumor growth and metastasis. J Natl Cancer Inst 48:1441-1455, 1972 SNODGRASS MJ, HANNA MG: Ultrastructural studies of histiocyte-tumor cell interactions during tumor regression after intralesional injection of Mycobacterium bovis. Cancer Res 33:701-716, 1973 MASTRANGELO MJ, KIM YH, BORNSTEIN RS, et al: Clinical and histiologic correlation of melanoma regression after intralesional BCG therapy: A case report. J Natl Cancer Inst 52:19-24, 1974 GUTTERMA:-I JU, MAVLlGIT GB, McBRIDE C, et al: Immuno· prophylaxis of malignant melanoma with systemic BCG: Study of strain, dose, and schedule. Natl Cancer Inst Monogr 39:205-212, 1973 PINSKY CM, HIRSHAUT Y, OETTGEN HF: Treatment of malignant melanoma by intratumoral injection of BCG. Nat! Cancer Inst Monogr 39:225-228, 1973 BORNSTEIN RS, MASTRANGELO MJ, SULlT H, et al: Immuno-
704
(21) (22) (23) (24)
MANGAN ET AL. therapy of melanoma with intralesional BCG. Natl Cancer Inst Monogr 39:213-220, 1973 KIN MOUTH JB, TAYLOR GW, HARPER RA: Lymphangiography technique for its clinical use in lower limbs. Br Med J 1:940-942, 1955 PRIER JE, ScHAFFER B, SKELLEY JE: Direct lymphangiography in the dog. J Am Vet Med Assoc 140:943-947, 1962 GISLER DB, BENSON RE, YOUNG RJ, et al: Colony husbandry of research monkeys. Ann NY Acad Sci 85:758-768, 1960 HILL WC: Primates: Comparative Anatomy and Taxonomy. VI. Cercopithecoidea. Edinburgh, Edinburgh Univ Press, 1966, p 84
(25) BAST RC, ZBAR B, BORSOS T, et al: BCG and cancer. Part 2.
N Engl J Med 290:1458-1469, 1974 (26) SPARKS FC, SILVERSTEIN MJ, HUNT JS, et al: Complications
of BCG immunotherapy in patients with cancer. N Engl J Med 289:827-830, 1973 (27) BLUMING AZ, VOGEL CL, ZIEGLER JL, et aI: Immunological effects of BCG in malignant melanoma: Two modes of administration compared. Ann Intern Med 76:405-411, 1972 (28) KLEIN E, HOLTERMANN OA, P APERMASTER B, et al: Immunologic approaches to various types of cancer with the use of BCG and purified protein derivatives. Nat! Cancer lnst Monogr 39:229-242, 1973
