Enhanced Neuroblastoma
Resection and Improved Survival in Murine (C1300-NB) After Preoperative Immunotherapy
By Carol L. Fowler, Stephen P. Brooks, Rely Squire, Gary A. Rich, Jon E. Rossman, Milton J. Finegold, James E. Allen, and Donald R. Cooney
Buffalo, New York and Houston, Texas 0 Advanced neuroblastoma treated with standard chemotherapy has a poor prognosis. Combination immunotherapy for murine neuroblastoma with retinyl palmitate, low-dose cyclophosphamide, and interleukin-2 resulted in increased survival, impaired tumor growth, easier surgical resection, and increased class I expression or tumor cells. Preoperative immunotherpay may be useful in treatment of advanced human neuroblastoma. Copyright o 7997 by W.B. Saunders Company INDEX WORDS: Neuroblastoma; immunotherapy; palmitate; cyclophosphamide; interleukin-2.
retinyl
S
URVIVAL of children with advanced neuroblastoma (NB) has not significantly improved in the past several decades. NB appears to be an immunogenic tumor. It has been postulated that immunotherapy might serve as an alternative or adjunctive mode of treatment and be more effective than standard chemotherapy or irradiation. Improved survival could potentially result from enhancement of host antitumor response. l-3Using the well-established experimental model of murine NB (C1300-NB) previous studies demonstrated that C1300-NB is immunogenic? Variable degrees of therapeutic response were achied with immunotherapy consisting of low-dose cyclophosphamide (CY) after surgical tumor debulking? as well as with interleukin-2Aymphokine activated killer cell (IL-2/LAK cell) therapy.6 In the present study, preoperative treatment of murine C1300-NB was evaluated using combinations of three immunomodulating agents: low-dose CY, IL-2, and retinyl palmitate (RP). The effect on survival, tumor growth, and histological characteristics of the tumor were studied. In addition, the effect of these immunotherapies on class I major histocompatibility complex (MHC) antigenie expression of the C1300-NB tumors was measured. Class I expression is an important consideration in immunotherapy becuase recognition and lysis of tumor cells by cytolytic T cells depends in part on the presence of class I molecules on the tumor cell surface.7 Both human and murine NB cell lines normally exhibit low class I expression, which may afford escape from host antitumor surveillance.58X9If class I expression could be upregulated by immunotherapy, antitumor cytolytic T cell activity might result in enhanced tumor regression.
JournalofPediatric
Surgery, Vol26, No 4 (April), 1991: pp 381-388
MATERIALS AND METHODS
Mice Female A/J mice, 8 to 10 weeks old, were obtained from Jackson Laboratories (Bar Harbor, ME). Animals were housed at a constant temperature and provided ad lib standard rodent chow and water.
Tumor Murine C1300-NB was maintained by serial passage both in vivo and in vitro. For passage in vivo, tumor cell suspensions were prepared by aspetically excising subcutaneous lesions. Tumors were mechanically disaggregated in phosphate buffered saline (PBS). After the viability of cells was determined by trypan blue exclusion, a suspension of 1 x 10” live tumor cells was injected subcutaneously in the flanks of syngeneic A/J mice.
Cyclophosphamide CY was obtained from Sigma Chemical Company (St Louis, MO). Each dose of CY was administered as a single intrapertioneal (IP) injection of either 25 mg,kg or 100 mg/kg on the days indicated in the treatment schedules (Fig 1).
Interleukin-2 Highly purified recombinant human IL-2 from Escherichia coli (Cetus Corporation, Emmeryville, CA) was reconstituted in sterile PBS to a dilution of 1.6 x 10’ IU/mL and injected intraperitoneally as O.l=mL (1.6 x 10’ IU).
Warnin A RP Aquasol A parenteral (retinyl palmitate, 50,000 IU/mL in water soluble form) was obtained fromArmor Pharmaceutical Co (Kankakee, IL). Doses of 0.05 mL (2,500 IU) were injected IP as indicated in the treatment schedules (Fig 1).
Treatment Schedules Forty-one mice with l.O-cm tumors (7 days after implantation) were divided into the following experimental groups: 1 untreated
From the Section of Pediatric Surgery Department of Surgery, School of Medicine and Biomedical Sciences, State University of New York, and the Children’s Hospital, Buffalo NY and the Department of Pathology, Texas Children S Hospital, Houston, TX. Supported in part by the Women & Children S Research Foundation of The Children’s Hospital of Buffalo, Inc, Buffalo, NY Presented at the 21st Annual Meeting of the American Pediam’c SurgicalAssociation, Vancouver, British Columbia, May 19-22, 1990. Address reprint requests to Donald R. Cooney, MD, Head, Section of Pediatric Surgery, Children S Hospital of Bufalo, 219 Bryant St, Buffalo, NY 14222. Copyright o 1991 by W.B. Saunders Company 0022-3468/91/2604-0005$03.0010
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Fig 1. Treatment and dose schedules. Preoperative immunotherapy with RP (2,500 IU twice a week), CY (day 2,100 mg/kg; day 9,25 mg/kg), IL-2 (1.6 x 1061U twice a day on days 4 to 9 and 11 to 13) was administered for 13 days to mice with l-cm C1300-NB tumors. Tumor resection was performed at 14 days.
control (n = 5); II, RP (n = 5); III, CY (n = 5); IV, IL-2 (n = 5); V, RP + CY (n = 5); VI, RP + IL-2 (n = 5); VII, CY + IL-2 (n = 5); and VIII, RP + CY + IL-2 (n = 6) (Fig 1). All treatment regimens were administered for 13 days. Tumor resections were performed in all groups on day 14. RP was given twice per week starting on day 1 of treatment (4 doses total). CY was administered 100 mg/kg on day 2, and 25 mgkg on day 9. IL-2 was injected twice per day with 1.6 x 10’ IU/dose on days 4 through 9 and 11 through 13.
Operative Procedure On day 14 of treatment, tumors were resected under sterile conditions using pentobarbital anesthesia. Tumor resections were performed as completely as possible and included local adjacent areas of involved skin, muscle, and peritoneum. Major resections of adjacent involved muscle were not performed.
Tumor Growth Daily measurements of tumor size were determined and recorded as “cm,z” the product of the tumor’s greatest width and length.
Histology Resected tumors were divided into two sections. Half of the specimen was fixed in formalin and submitted for histological examination. The other half was analyzed for class I antigenic expression.
MHC Class I Expression Some resected tumors were analyzed as whole specimens for class I expression. Other tumors were separated into specimens from areasthat grossly appeared to be either nectrotic (avascular and white) or viable (pink and homogeneous). Resected tumors were manually disaggregated in PBS. Cell suspensions containing 10’ cells/ 0.05 mL, including both viable and nonviable cells, were incubated on ice with anti-H, monoclonal antibody (Hybirtech, San Diego, CA) for 30 minutes. After washing with PBS, the cells were incubated on ice with fluorescein isotheocyanate-conjugated second antibody for 30 minutes before two final washes with PBS. Flow cytometric analysis was determined with EPICS (Coulter Corp, Hialeah, FL). Class I expression was reported as mean fluorescence.
Serum Vitamin A Levels Using a modification of methods reported by Neeld and Pearson”’ and Tietz,” serum vitamin A levels were determined prior to treatment and on day 13 of therapy for experimental groups
receiving RP. All serum samples, solutions, and procedures were protected from light. Vitamin A and B carotene standards were prepared as per published methods.“~” From each group of animals 1.0 to 1.5 mL of retroorbital blood was pooled and the serum was separated by centrifugation. In a glass-stoppered centrifuge tube, 0.5 to 0.75 mL of serum was added to 1.0 mL of 95% ethanol, and vortexed. Petroleum ether (0.8 mL) was added, then the sample was vortexed for 7 minutes. After centrifugation, 0.6 mL of the upper ether phase was transferred to a cuvet. The absorbance was immediately read at 450 nm against an ether blank. The contents of the cuvet were then evaporated to dryness in a 50°C water bath with the aid of a fine stream of nitrogen. Chloroform (0.05 mL) was added to each cuvet to dissolve the residue. TFA: chloroform (1:2 parts) solution (0.5 mL) was added, mixed briefly, then immediately read at 620 nm against a trifluoroaacetic acid solution blank. Vitamin A (free alcohol) ug/dL = Abs,
- (Abs,,, x F) F x g
x 100 x .872
Where F = B-carotene correction factor obtained from p-carotene, standard curve = 3.12 for this laboratory; 0.8 = mL of ether added to serum; 0.6 = mL of ether extract used for assay; 100 = conversion of retinyl acetate ug/mL to ug/dL, .872 = correction factor for use of retinyl acetate instead of retinol as the standard; this is the ratio of molecular weight of retinol to retinyl acetate.
Statistical Methods Survival data were compared with log-rank test. Other data are presented as group mean ? SEM. ANOVA and Duncan multiple comparison tests were used for group comparisons. Paired, twotailed Student’s r test was used to compared class I expression within tumor sections. P values of 5 0.05 were considered significant. RESULTS
Survival
As compared with the untreated control group, all four groups receiving IL-2 treatment (groups IV, VI, VII, and VIII) demonstrated statistically prolonged survivals (P < .005). The longest survival and the only two tumor cures (33%) were noted in treatment group VIII. Survival in groups II (RP), III (CY), and V (RP + CY) did not offer statistically from the control animals, although the survival of animals in group III approached significance (Figs 2A and 2B). Tumor Growth Rate
Initial tumor growth was similar in all gruops until day 5 of treatment, when a divergence of growth rates was observed. This corresponded to 1 day after IL-2 treatment was begun. By day 14, the most striking inhibition of tumor growth was noted in group VII (RP + CY + IL-2) in which no significant tumor growth occurred after day 5. On day 14 tumors from group VIII were significantly smaller than all other groups (3.31 ~fr0.36 cm’, P < 0.001) except group
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The gross appearance of the cut sections of resected tumors varied depending on the treatment regimen. The usual appearance of advanced control C1300-NB is that of a large irregular mass that has central areas of hemorrhagic necrosis. A striking difference was noted in tumors treated with IL-2, especially from treatment groups VIII and VIII (Fig 4). All of these tumors were small and contained large areas of coagulation necrosis, which appeared as homeogeneous light-colored tissue or as heterogeneous areas of white nodules and/or bands interdispersed within areas of viable tissue. No hemorrhagic necrosis was observed in these treated tumors. Marginal rims of viable tumor were occasionally noted in these lesions. histological heterogeneity was also found in the majority of the resected tumors from treatment groups IV (IL-2) and VI (RP + IL-2). The viable tumor cells at the periphery of the successfully
A 6-
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--g RPGY+ILP Fig 2. Survival. (A, B) Length of survival is noted after various combinations of preoperative immunotherapy were administered to mice with C1300-NB. Survival of all groups receiving IL-2 (IL-2, RF + IL-2, CY + IL-2, and RP + CY + k-2) was significantly prolonged compared with untreated controls fP < ,005). The longest survival was noted in the group treated with RP + CY + IL-2.
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VII (CY + IL-2). Tumors from CY + IL-2-treated animals measured 4.78 + 0.20 cm2 and were also significantly smaller than all groups (P < .OOl) except the RP + CY + IL-Ztreated mice (Figs 3A and 3B). Tumor Appearance and Histology
In the two treatment groups demonstrating the most profound effect on arresting tumor growth (RP + CY + IL-2 and CY + IL-2), all lesions were small and easily resectable at day 14 of therapy. In most of these animals, inflammatory process produced adherence of the tumor to the overlying sking. Otherwise, these tumors appeared encapsulated and did not grossly invade the underlying muscle. IL-2- and RP + IL-Ztreated mice survived until day 14 to undergo resection, but had extremely large tumors. None of these large tumors could be entirely extirpated and rapidly recurred after resection. Only a few mice from groups II (RP) and I (control) survived to undergo surgery. Their tumors were very large and not completely resectable.
“E 0
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Fig 3. Tumor growth rate. (A) single-agent therapies. No significant difference in tumor growth was observed over a ICday treatment period. (B) Tumor growth in all multiagent therapy groups was similar until day 5 of treatment. One day after IL-2 was begun [day 4). a divergence of growth rates was observed. At day 14, tumors from the group RP + CY + IL-2 were significantly smaller than those from the other groups (P < ,001). Lesions from CY + IL-2-treated mice were also statistically smaller than tumors from all groups except RP + CY + IL-2.
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s
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Fig 5. Class I expression of tumors from treatment groups as measured by mean fluorescence. A trend toward increased class I expression was noted in the groups that received IL-2. RP + CY + IL-2-treated animals exhibited statistically greater class I expression than all non-IL-2 groups and RP + IL-2-treated mice (P < .0035).
higher than the viable areas of resected tumor, which averaged 311.17 ? 24.07 (P = .0029) (Fig 6). Fig 4. Tumor appearance. C1300-NB tumors were resected at day 14 of treatment. Tumor seen at the top of the figure is from an untreated animal. Note the contrast between this large, friable hemorrhagic tumor and the small, fibrotic tumor below that was resected from a mouse treated with RP + CY + IL-2
treated nodules were no less pleomorphic or mitotitally active than those in the continually growing tumors. There were no significant lymphocytic infiltrates in any of the tumors. Tumors from group VIII (RP + CY + IL-2) appeared to be completely excised at operation. However 4 of 6 lesions recurred postoperatively and resulted in death of the animals within 13 to 22 days. Postoperative survival was significantly longer (P < .OOl) in this group than the other experimental groups in which recurrent tumors proved to be lethal in 4 to 16 days following surgery. Incomplete resections that left gross residual tumor resulted in tumorassociated deaths in 2 to 9 days. Class I Expression
As measured by mean fluorescence, there was a trend of higher class I expression in tumors from all groups treated with IL-2. Tumors from RP + CY + IL-2-treated mice measured 443.0 + 26.6, cm’, which was significantly higher than all other groups (P = .035), except groups VII (CY + IL-2; 400 2 47.4 cm’) (Fig 5). Histologically heterogeneous tumors exhibited both whitish necrotic and pink viable areas on cut section. These two separate areas were compared histologically in 7 tumors. The class I expression of the areas of coagulation necrosis measured 478.39 _+ 24.17 and was consistently and significantly
Vitamin A Levels
Serum vitamin A levels or normal, untreated A/J mice was 15 to 20 l.q$dL. In mice treated with RP, the serum level was elevated to a clinically nontoxic level of 25 to 35 Fg/dL. 600
I
P)
G 3 ii
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OF
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Fig 6. Class I expression from different areas of the same tumor; sections of the tumor that appeared whiie and fibrotic were compared with areas that appeared to be viable. Significantly increased class I expression was noted in the fibrotic areas (P < .OOzS).
PREOPERATIVE IMMUNOTHERAPY
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DISCUSSION The rationale for evaluating the three immunotherapeutic agents of low-dose CY, IL-2, and RP stems from previous studies. In a murine model of advanced C1300-NB, therapeutic responses were produced with surgical debulking of the tumor followed by immunomodulating doses of CY.’ The proposed mechanism of action of low-dose CY involves eradication of tumor-enhanced systemic T suppressor cell function. It was postulated that a CY-induced reduction in T suppressor activity might be synergistic with the effects of other immune modulators such as IL-2 and RP. Previous studies had determined that treatment with IL-2 alone was effective in improving survival of mice with IP C1300-NB.’ Retinoids were studied because of their demonstrated antineoplastic effects in a variety of other tumor models.12~‘5The mechanism of action of retinoids includes both tumoricidal and tumoristatic effects, immunomodulation, and cellular differentiation and maturation of NB cell lines.9*‘6Furthermore, synergistic antineoplastic effects of retinoids combined with CY,“-” and CY combined with IL-22@23 have been reported. In the present study, preoperative triple immunotherapy using CY, IL-2, and RP resulted in the best survival rate and the greatest inhibition of C1300-NB tumor growth when compared with all single- and double-drug regimens. A proposed mechanism by which this apparent synergism occurs is as follows. Previous studies demonstrated that the therapeutic effect of CY in the C1300-NB model involves eradication of tumor-enhanced T suppressor cell function.’ Moreover, it has been noted that CY-sensitive T suppressor cells can produce IL-2 inhibitors that neutralize the biological activity of IL-2.” Therefore, the observed synergism of CY and IL-2 may occur through CY-induced down-regulation of T suppressor cell function and reduction of IL-2 inhibitors, each resulting in elevated antitumor IL-2 activity. This may explain both the enhanced therapeutic effect of combined CY and IL-2 and the effectiveness of low doses of IL-2 when combined with CY therapy. The low dose of IL-2 used in the present study, 3.2 x lo5 III/d, was effective against C1300-NB and drug toxicity was limited. Toxicity-induced anorexia and lethargy were reversed by withholding therapy for 1 day after 6 days of therapy. As previously noted, the synergistic effects of CY combined with IL-2,?“-23 retinoids combined with CY,‘7-‘9and retinoids plus IL-2” have been studied. In combination these three immunotherapeutic agents may allow the IL-2 dose to be decreased even further. The use of nontoxic low doses of IL-2 constitutes a significant advantage over the high-dose IL-2 regimens currently being evalu-
305
ated in most human clinical trials. Severe capillary leak syndrome is a common complication of highdose IL-2 therapy, and unfortunately, has been accompanied by occasional mortality.” The addition of RP to CY + IL-2 therapy improved survival and further inhibited tumor growth. Vitamin A analogs have long been shown to demonstrate tumoricical and tumoristatic properties.‘“‘5 It is significant to note that their effectiveness in the treatment of NB has not yet been reported. The antitumor mechanism of retinoids is not well-defined. Immune potentiation by retinoids has been reported and includes increased accessory cell (macrophage and dendritic cells) functionn*% and increased Lyt-1 subset of mouse T lymphocyte population.‘2.29 Increased IL-2 production by spleen cells has also been observed in mice treated with vitamin A-acetate.z Direct cytotoxicity from retinoids may also occur from destabilization of cell membranes and release of lysosomal enzymes resulting in cell lysis.” Experimentally, additive antitumor effects have been noted when retinoid therapy is combined with radiation of chemotherapeutic agents, including CY.15.‘7-‘9 NB is similar to other malignant tumors of epithelial origin in that they exhibit low levels of class I MHC antigen (MHC I) on their cell surface.‘.’ Expression of MHC I antigen is a prerequisite for host cytolytic T cell (CTC) recognition and lysis of aberrant cells.’ The effectiveness of immunotherapy for NB may be limited by this phenomenon. If class I expression of NB could be upregulated, making tumors a better CTC target, immunotherapy may be more effective. Analysis of C1300-NB tumors for class I expression showed that the experimental group treated with RP + CY + IL-2 demonstrated the highest class I expression. Because this expression was significantly higher than that of untreated tumors, in vivo upregulation of class I expression appears to be possible. In fact, class I expression of all groups receiving IL-2 was significantly greater than the other experimental groups. Analysis of the viable and nonviable sections of treated tumors showed that class I expression was significantly increased in the areas that showed a pronounced therapeutic effect, as evidenced by the presence of coagulation necrosis. These conspicuous areas of coagulation necrosis weren noted only in the treatment groups that received IL-2. Up-regulated class I expression may have resulted from IL-2-induced autologous interferon activity as suggested by other studies.30 In experimental murine’ and human9 NB cell lines, class I expression has been increased by treatment with interferon. RP may have augmented these effects. It is an agent
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that has been shown to induce cellular antigenic modulation.9.‘6 and the morphological differentiation3”” of NB cells in culture. The RP + CY + IL-2 treatment group exhibited the best therapeutic response as well as the greatest class I expression. This observation reinforces the importance of immunotherapy-induced class I expression. In a study of histological specimens from children with NB, Squire et al noted the greatest class I expression in stage IV-S NB.’ IV-S NB is unique in that these lesions exhibit a remarkable tendency for spontaneous regression. This phenomenon may represent a type of host antitumor immune response similar to that observed in the present experimental model. In our current study, the duration of preoperative treatment was selected as 14 days because pilot studies demonstrated that the inhibitory effect on tujor growth was usually transient. RP + CY + IL-Ztreated tumors usually began increasing in size at 12 to 14 days of treatment. The resurgance of tumor growth both prior to or after resection may represent proliferation of resistance clones of cells within the lesion. Alternatively, tumor recurrence may be due to growth within areas that were not effectively treated because adequate doses of the therapeutic agents did not reach the lesion. Tumor regression induced by immunotherapy may be due to several factors. Whereas regression at the periphery of the tumor is probably dependent on host immunity, central hemorrhagic necrosis does not necessarily depend on immunologic factors.33 The similarity of the pattern of tumor necrosis induced by immunotherapy with RP + CY + IL-2 to that reported using other agents may be a clue to the mechanism of action. These histological changes are similar to those induced by interferon and tumor necrosis factor. In particular, the coagulation necrosis observed in the present study is very similar to the response produced by interferon as reported by Dvorak and Gresser.34 The pattern of tumor necrosis is also similar to that noted in mouse sarcomas treated by tumor necrosis factor, in which a viable rim
of tumor was noted peripheral to the area of central hemorrhagic necrosis.34 An interesting observation of this study was the fact that the admixture of irregular areas of viable tumor and necrosis were noted primarily within the RP + CY + IL-2- and CY + IL-2-treated tumors. One of the most significant concerns relating to the adequacy of therapy is the necessity of delivering therapeutic agents to the lesion in tumoricidal doses. Some areas within the tumor may be less vascular than normal tissue. Transport of chemotherapeutic agents into the tumor may be also reduced due to high intratumor extracellular fluid pressure. Circulation of antineoplastic agents within the tumor interstitium may also be limited by the large interstitial space and loss of fluid from the tumor’s periphery.35 In addition, the physical properties restricting delivery of traditional systemic chemotherapeutic agents probably depends on the size of the tumor. It may indeed be beneficial to reduce a large primary tumor into several smaller noncontiguous lesions separated by necrotic areas, as was observed in this study. The resulting residual nests of tumor tissue may possibly be more effectively treated with systemic agents. This study suggests that a combination of biological response modifiers administered at low nontoxic doses may be an effective component of multimode1 therapy. However, it is important to note that despite the initial antitumor response to combination immunotherapy, postoperative recurrences were frequent in all experimental groups. It appears that an modified regimen or additional postoperative therapy will be needed to improve survival. On the other hand, these agents seem to restore the balance of immunologic responsiveness in favor of the host. Most significant is the fact that these regimens might be used prior to operation without suppressing the patient’s immune defense or causing other serious complications commonly associated with standard chemotherapy or irradiation. Such well-tolerated therapeutic programs potentially could be used as maintenance therapy between pulses of standard chemotherapy following operation.
REFERENCES 1. Squire R, Fowler CL, Brooks SP, et al: The relationship of class I MHC antigen expression to stage IV-S disease and survival of neuroblastoma. J Pediatr Surg 25:381-386,199O 2. Sigal RK, Reynolds JV, Markmann JF, et al: Upregulation of MHC class I: Effect on growth and LAK sensitivity of neuroblastoma. Surg Forum 39:572-575,1988 3. Necheles TF, Tefft M, Weinberg V: Randomized trial of immunotherapy in the treatment of advanced neuroblastoma, in Terry WD, Rosenberg SA (eds): Immunotherapy of Human Cancer. New York, NY, Elsevier, 1982, pp 377-383
4. Bruce J, Brooks SP, Rich GA, et al: Effects of immunostimulation on host survival in A/J mice with transplantable Cl300 neuroblastoma. Curr Surg 45:17-19,1988 5. Fowler CL, Brooks SP, Rossman JE, et al: Postoperative immunotherapy of murine C1300-neuroblastoma. J Pediatr Surg 25:229-237, 1990 6. Brooks SP, Cooney DR, Fowler CL: Treatment of murine Cl300 neuroblastoma with IL-2/LAK cell therapy. Presented at the meeting of The Association for Academic Surgery, Salt Lake City, UT, November 16-19, 1988
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7. Tanaka K, Yoshioka T, Bieberich C, et al: Role of the major histocompatibility complex class I antigens in tumor growth and metastases. Annu Rev Immunol6:359-380, 1988 8. Lampson LA, Fisher LA, Whelan JP: Striking paucity of HLA-A. B, C and 82-microglobulin of human neuroblastoma cell lines. J Immunol130:2471-2478,1983 9. Gross N, Beck D, Favre S, et al: In vitro antigenic modulation of human neuroblastoma cells induced by IFN-gamma, retinoic acid and dibutyryl cyclic AMP. Int J Cancer 39:521-529,1987 10. Neeld JF Jr, Pearson WN: Macro- and micromethods for the determination of serum vitamin A using trifluoroacetic acid. J Nutr 79~454462, 1963 11. McCormick, DB: Vitamins, in Tietz NW (ed): Fundamentals of Clinical Chemistry. Philadelphia, PA, Saunders, 1986, pp 927-964 12. Eccles SA: Effects of retinoids on growth and dissemination of malignant tumors: Immunological considerations. Biochem Pharm 34:1599-1610,1985 13. Felix EL, Cohen MH, Loyd BC: Immune and toxic antitumor effects of systemic and intralesional vitamin A. J Surg Res 21:307-312, 1976 14. Watson RR, Moriguchi S, Gensler HL: Effects of dietary retinyl palmitate and selenium on tumoricidal capacity of macrophages in mice undergoing tumor promotion. Cancer Lett 36:181187.1987 15. Seifter E, Rettura G, Padawer J, et al: Regesssion of C3HBA mouse tumor due to x-ray therapy combined with supplemental B-carotene ofvitamin A. JNCI 71:409-417,1983 16. Reynolds CP, Maples J: Modulation of cell surface antigens accompanies morphologic differentiation of human neuroblastoma cell lines. Prog Clin Biol Res 17513-37, 1985 17. Nathanson L, Maddock CL, Hall TC: Exploratory studies of vitamin A and cyclophospbamide in tumor-bearing mice. J Clin Pharm 9:359-373,1969 18. Levenson SM, Rettura G, Seifter E: Effects of supplemental dietary vitamin A and B-carotene on experimental tumors, local tumor excision, chemotherapy, radiation injury, and radiotherapy, in Butterworth CE Jr, Hutchinson ML (eds): Nutritional Factors in the Induction and Maintenance of Malignancy. New York, NY, Academic, 1983, pp 169-203 19. Cohen MH, Carbone PP: Enhancement of the antitumor effects of 1,3-bis (2-chloroethyl)-1-nitrosourea and cyclophosphamide by vitamin A. JNCI 48:921-926,1972 20. Lee K, O’Donnell RW, Marquis D, et al: Eradication of palpable intradermal murine bladder tumors by systemic interleukin-2 and cyclophosphamide in C3H mice. J Biol Response Mod 7:32-42,1988 21. Hosokawa M, Yabiku T, Ikeda J, et al: Effects of a combination of cyclophosphamide and human recombinant inter-
leukin-2 on pulmonary metastases after the surgical removal of a 3-methylcholanthrene-induced primary tumor in autochthonous mice. Jpn J Cancer Res 79:1147-1154,1988 22. Kolitz JE, Wong GY, Welte K, et al: Phase I trial of recombinant interleukin-2 and cyclophosphamide: Augmentation of cellular immunity and T-cell mitogenic response with long-term administration of rIL-2. J Biol Response Mod 7:457-472. 1988 23. Topalian SL, Solomon D, Avis FP, et al: Immunotherapy of patients with advanced cancer using tumor-infiltrating lymphocytes and recombinant interluekin-2: A pilot study. J Clin Oncol 6:839853.1988 24. Kucharz El, Goodwin JS: Minireview: Serum inhibitors of interleukin-2. Life Sci 42:1485-1491, 1988 25. Colizzi V, Malkovsky M: Augmentation of interleukin-2 production and delayed hypersensitivity in mice infected with mycobacterium bovis and fed a diet supplemented with vitamin A acetate. Infect Immunol48:581-583, 1985 26. Rosenberg SA, Lotze MT, Yang JC, et al: Experience with the use of high-dose interleukin-2 in the treatment of 652 cancer patients. Ann Surg 210:474-485, 1989 27. Moriguchi S, Werner L, Watson RR: High dietary vitamin A (retinyl palmitate) and cellular immune functions in mice. Immunology 56:169-177,1985 28. Katz DR, Krzymala M, Turton JA, et al: Regulation of accessory cell function by retinoids in murine immune responses. Br J Exp Path01 68:343-350,1987 29. Prabhala RH, Maxey V, Hicks MJ, et al: Enhancement of the expression of activation markers on human peripheral blood mononuclear cells by in vitro culture with retinoids and carotenoids. J Leukoc Biol45:249-254,1989 30. Merigan TC: Is recombinant interleukin-2 the best way to deliver interferon-gamma in human disease? J Interferon Res 7:635-639,198J 31. Shea TB, Fischer I, Sapirstein VS: Effect of retinoic acid on growth and morphologic differentiation of mouse NB2a neuroblastoma cells in culture. Dev Brain Res 21:307-314, 1985 32. Side11N: Retinoic acid-induced growth inhibition and morphologic differentiation of human neuroblastoma cells in vitro. JNCI 68:589-592, 1982 33. North RJ, Have11 EA: The antitumor function of tumor necrosis factor (TNF):II. Analysis of the role of endogenous TNF in endotoxin-induced hemorrhagic necrosis and regression of an established sarcoma. J Exp Med 167:1086-1099,1988 34. Dvorak HF, Gresser I: Microvascular injury in pathogenesis of interferon-induced necrosis of subcutaneous tumors in mice. JNCI 81:497-502,1989 35. Jain RK: Delivery of novel therapeutic agents in tumors: Physiological barriers and strategies. JNCI 81:570-576, 1989
Discussion G. Haase (Denver, CO): The authors have presented an elegant laboratory study evaluating preoperative immunotherapy in the murine Cl300 neuroblastoma model. It is a reasonable postulate that these three agents in combination could inhibit tumor growth and improve survival. Although the classic experimental model used is immunogenic, its use comprises the study. Why was one of several well-
established human neuroblastoma cell lines not used? The impact of the biological response modifiers might then have been more easily transferred to the clinical setting. I am also concerned that recombinant human IL-2 was used. What effect does this human product, which may function by a different mechanism, have on a murine cell line? The biggest impact on tumor
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growth and survival occurred in experimental groups receiving the IL-2, so the validity of these observations is under question. The authors conclude that a combination of these agents administered at low doses would be part of an effective human therapeutic regimen. This is contrary to current oncologic thought (the Coldie-Goldman hypothesis) that antitumor therapy should be given intensely, up-front, and in the highest possible does consistent with cell cycle kinetics followed by a rescue technique such as bone marrow transplantation. With this current clinical emphasis on dose intensity, how important do the authors think their experimental results really are? C. Fowler (response): Dr. Haase, the reason we used the murine model is that we have had experience with it for several years. The reason we don’t at present evaluate immunotherapy on human cell lines is that although there are numerous human cell lines available, we were trying to establish an in vivo model because obviously the effects of immunotherapy will be different in culture and in vivo. Human interleukin was used because it is readily available and immunologically, as far as we know, it acts the same as a mouse interleukin. The low doses we used are contrary to what the oncologists use. Oncologists teach that “more is better,” but when you see patients with numerous toxic side effects, it is apparent that lowdose immunotherapy should provide some advantage over high-dose chemotherapy because the low doses we were using were nontoxic. Using nontoxic low doses to enhance the patient’s own host immune defense should be better than completely immunocompromising a patient with high-dose therapy and then rescuing him/her with other drugs. This is especially the case in neuroblastomas, because standard chemotherapy has not been shown to satisfactorily improve the prognosis of children with advanced disease. R.M. Filler (Toronto, Ontario): Could you perhaps theorize why this works that way? Is there any indication that the resistance of the cells to chemotherapy is changed? C. Fowler (response): In this paper, we haven’t gone into the mechanisms of action. Earlier, we showed in this model that low-dose cyclophosphamide in the
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doses that we use presently decreases an increased T-suppressor cell activity that Cl300 neuroblastoma induces. Vitamin A in the literature has been shown to be tumorcidal and tumoristatic. It has increased accessory cell function and what is especially interesting in pertaining to neuroblastoma is that if you put any one of the retinoids in culture with either human or mouse neuroblastoma, it will produce a differentiation and maturation of the cells. That’s only been seen in vitro not in vivo, but possibly in our in vivo Cl300 neuroblastoma model, retinoids could enhance some of the other immunotherapies that we are using to produce this same effect. IL-2 is a lymphokine that activates the other T cells. Other papers have been written about synergism between vitamin A and IL-2 as well as vitamin A and cyclophosphamide. Vitamin A increases cell membrane labilization so that agents such as cyclophosphamide can enter the tumor cell more easily and produce their effects. Qclophosphamide and IL-2 also have been shown to be synergistic. Cyclophosphamide decreases IL-2 inhibitors produced by T suppressor cells, thus enhancing IL-2 effects. The histological changes that we’ve seen on cut specimens of treated tumors included coagulation necrosis and rims of viable tumor. In the literature interferon produces some of these effects, so perhaps interferon was induced as part of the immunotherapy. This is further supported in that interferon is known to increase class I expression and increased class I expression was also noted in the treated tumors. Finally, tumor necrosis factor is another possible mechanism. It produces hemorrhagic necrosis and a viable rim of tumor, a pattern that we have seen in some of these tumors. Our next experiments will be to see exactly which mechanisms are used in among all these possibilities. D. Tapper (Seattle, WA): Dr Fowler, can I ask you two things. Is your neuroblastoma model a nonmetastasizing logo tumor model? Is that correct? The purpose then, unlike the other IL-2 studies, is to measure logo changes in the tumor? To go back to what Dr Haase asked, do you know the genetic make-up of this tumor? C. Fowler (response): The clone we have is nonmetastasizing. No.
Resection and Improved Survival in Murine (C1300-NB) After Preoperative Immunotherapy
By Carol L. Fowler, Stephen P. Brooks, Rely Squire, Gary A. Rich, Jon E. Rossman, Milton J. Finegold, James E. Allen, and Donald R. Cooney
Buffalo, New York and Houston, Texas 0 Advanced neuroblastoma treated with standard chemotherapy has a poor prognosis. Combination immunotherapy for murine neuroblastoma with retinyl palmitate, low-dose cyclophosphamide, and interleukin-2 resulted in increased survival, impaired tumor growth, easier surgical resection, and increased class I expression or tumor cells. Preoperative immunotherpay may be useful in treatment of advanced human neuroblastoma. Copyright o 7997 by W.B. Saunders Company INDEX WORDS: Neuroblastoma; immunotherapy; palmitate; cyclophosphamide; interleukin-2.
retinyl
S
URVIVAL of children with advanced neuroblastoma (NB) has not significantly improved in the past several decades. NB appears to be an immunogenic tumor. It has been postulated that immunotherapy might serve as an alternative or adjunctive mode of treatment and be more effective than standard chemotherapy or irradiation. Improved survival could potentially result from enhancement of host antitumor response. l-3Using the well-established experimental model of murine NB (C1300-NB) previous studies demonstrated that C1300-NB is immunogenic? Variable degrees of therapeutic response were achied with immunotherapy consisting of low-dose cyclophosphamide (CY) after surgical tumor debulking? as well as with interleukin-2Aymphokine activated killer cell (IL-2/LAK cell) therapy.6 In the present study, preoperative treatment of murine C1300-NB was evaluated using combinations of three immunomodulating agents: low-dose CY, IL-2, and retinyl palmitate (RP). The effect on survival, tumor growth, and histological characteristics of the tumor were studied. In addition, the effect of these immunotherapies on class I major histocompatibility complex (MHC) antigenie expression of the C1300-NB tumors was measured. Class I expression is an important consideration in immunotherapy becuase recognition and lysis of tumor cells by cytolytic T cells depends in part on the presence of class I molecules on the tumor cell surface.7 Both human and murine NB cell lines normally exhibit low class I expression, which may afford escape from host antitumor surveillance.58X9If class I expression could be upregulated by immunotherapy, antitumor cytolytic T cell activity might result in enhanced tumor regression.
JournalofPediatric
Surgery, Vol26, No 4 (April), 1991: pp 381-388
MATERIALS AND METHODS
Mice Female A/J mice, 8 to 10 weeks old, were obtained from Jackson Laboratories (Bar Harbor, ME). Animals were housed at a constant temperature and provided ad lib standard rodent chow and water.
Tumor Murine C1300-NB was maintained by serial passage both in vivo and in vitro. For passage in vivo, tumor cell suspensions were prepared by aspetically excising subcutaneous lesions. Tumors were mechanically disaggregated in phosphate buffered saline (PBS). After the viability of cells was determined by trypan blue exclusion, a suspension of 1 x 10” live tumor cells was injected subcutaneously in the flanks of syngeneic A/J mice.
Cyclophosphamide CY was obtained from Sigma Chemical Company (St Louis, MO). Each dose of CY was administered as a single intrapertioneal (IP) injection of either 25 mg,kg or 100 mg/kg on the days indicated in the treatment schedules (Fig 1).
Interleukin-2 Highly purified recombinant human IL-2 from Escherichia coli (Cetus Corporation, Emmeryville, CA) was reconstituted in sterile PBS to a dilution of 1.6 x 10’ IU/mL and injected intraperitoneally as O.l=mL (1.6 x 10’ IU).
Warnin A RP Aquasol A parenteral (retinyl palmitate, 50,000 IU/mL in water soluble form) was obtained fromArmor Pharmaceutical Co (Kankakee, IL). Doses of 0.05 mL (2,500 IU) were injected IP as indicated in the treatment schedules (Fig 1).
Treatment Schedules Forty-one mice with l.O-cm tumors (7 days after implantation) were divided into the following experimental groups: 1 untreated
From the Section of Pediatric Surgery Department of Surgery, School of Medicine and Biomedical Sciences, State University of New York, and the Children’s Hospital, Buffalo NY and the Department of Pathology, Texas Children S Hospital, Houston, TX. Supported in part by the Women & Children S Research Foundation of The Children’s Hospital of Buffalo, Inc, Buffalo, NY Presented at the 21st Annual Meeting of the American Pediam’c SurgicalAssociation, Vancouver, British Columbia, May 19-22, 1990. Address reprint requests to Donald R. Cooney, MD, Head, Section of Pediatric Surgery, Children S Hospital of Bufalo, 219 Bryant St, Buffalo, NY 14222. Copyright o 1991 by W.B. Saunders Company 0022-3468/91/2604-0005$03.0010
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Fig 1. Treatment and dose schedules. Preoperative immunotherapy with RP (2,500 IU twice a week), CY (day 2,100 mg/kg; day 9,25 mg/kg), IL-2 (1.6 x 1061U twice a day on days 4 to 9 and 11 to 13) was administered for 13 days to mice with l-cm C1300-NB tumors. Tumor resection was performed at 14 days.
control (n = 5); II, RP (n = 5); III, CY (n = 5); IV, IL-2 (n = 5); V, RP + CY (n = 5); VI, RP + IL-2 (n = 5); VII, CY + IL-2 (n = 5); and VIII, RP + CY + IL-2 (n = 6) (Fig 1). All treatment regimens were administered for 13 days. Tumor resections were performed in all groups on day 14. RP was given twice per week starting on day 1 of treatment (4 doses total). CY was administered 100 mg/kg on day 2, and 25 mgkg on day 9. IL-2 was injected twice per day with 1.6 x 10’ IU/dose on days 4 through 9 and 11 through 13.
Operative Procedure On day 14 of treatment, tumors were resected under sterile conditions using pentobarbital anesthesia. Tumor resections were performed as completely as possible and included local adjacent areas of involved skin, muscle, and peritoneum. Major resections of adjacent involved muscle were not performed.
Tumor Growth Daily measurements of tumor size were determined and recorded as “cm,z” the product of the tumor’s greatest width and length.
Histology Resected tumors were divided into two sections. Half of the specimen was fixed in formalin and submitted for histological examination. The other half was analyzed for class I antigenic expression.
MHC Class I Expression Some resected tumors were analyzed as whole specimens for class I expression. Other tumors were separated into specimens from areasthat grossly appeared to be either nectrotic (avascular and white) or viable (pink and homogeneous). Resected tumors were manually disaggregated in PBS. Cell suspensions containing 10’ cells/ 0.05 mL, including both viable and nonviable cells, were incubated on ice with anti-H, monoclonal antibody (Hybirtech, San Diego, CA) for 30 minutes. After washing with PBS, the cells were incubated on ice with fluorescein isotheocyanate-conjugated second antibody for 30 minutes before two final washes with PBS. Flow cytometric analysis was determined with EPICS (Coulter Corp, Hialeah, FL). Class I expression was reported as mean fluorescence.
Serum Vitamin A Levels Using a modification of methods reported by Neeld and Pearson”’ and Tietz,” serum vitamin A levels were determined prior to treatment and on day 13 of therapy for experimental groups
receiving RP. All serum samples, solutions, and procedures were protected from light. Vitamin A and B carotene standards were prepared as per published methods.“~” From each group of animals 1.0 to 1.5 mL of retroorbital blood was pooled and the serum was separated by centrifugation. In a glass-stoppered centrifuge tube, 0.5 to 0.75 mL of serum was added to 1.0 mL of 95% ethanol, and vortexed. Petroleum ether (0.8 mL) was added, then the sample was vortexed for 7 minutes. After centrifugation, 0.6 mL of the upper ether phase was transferred to a cuvet. The absorbance was immediately read at 450 nm against an ether blank. The contents of the cuvet were then evaporated to dryness in a 50°C water bath with the aid of a fine stream of nitrogen. Chloroform (0.05 mL) was added to each cuvet to dissolve the residue. TFA: chloroform (1:2 parts) solution (0.5 mL) was added, mixed briefly, then immediately read at 620 nm against a trifluoroaacetic acid solution blank. Vitamin A (free alcohol) ug/dL = Abs,
- (Abs,,, x F) F x g
x 100 x .872
Where F = B-carotene correction factor obtained from p-carotene, standard curve = 3.12 for this laboratory; 0.8 = mL of ether added to serum; 0.6 = mL of ether extract used for assay; 100 = conversion of retinyl acetate ug/mL to ug/dL, .872 = correction factor for use of retinyl acetate instead of retinol as the standard; this is the ratio of molecular weight of retinol to retinyl acetate.
Statistical Methods Survival data were compared with log-rank test. Other data are presented as group mean ? SEM. ANOVA and Duncan multiple comparison tests were used for group comparisons. Paired, twotailed Student’s r test was used to compared class I expression within tumor sections. P values of 5 0.05 were considered significant. RESULTS
Survival
As compared with the untreated control group, all four groups receiving IL-2 treatment (groups IV, VI, VII, and VIII) demonstrated statistically prolonged survivals (P < .005). The longest survival and the only two tumor cures (33%) were noted in treatment group VIII. Survival in groups II (RP), III (CY), and V (RP + CY) did not offer statistically from the control animals, although the survival of animals in group III approached significance (Figs 2A and 2B). Tumor Growth Rate
Initial tumor growth was similar in all gruops until day 5 of treatment, when a divergence of growth rates was observed. This corresponded to 1 day after IL-2 treatment was begun. By day 14, the most striking inhibition of tumor growth was noted in group VII (RP + CY + IL-2) in which no significant tumor growth occurred after day 5. On day 14 tumors from group VIII were significantly smaller than all other groups (3.31 ~fr0.36 cm’, P < 0.001) except group
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The gross appearance of the cut sections of resected tumors varied depending on the treatment regimen. The usual appearance of advanced control C1300-NB is that of a large irregular mass that has central areas of hemorrhagic necrosis. A striking difference was noted in tumors treated with IL-2, especially from treatment groups VIII and VIII (Fig 4). All of these tumors were small and contained large areas of coagulation necrosis, which appeared as homeogeneous light-colored tissue or as heterogeneous areas of white nodules and/or bands interdispersed within areas of viable tissue. No hemorrhagic necrosis was observed in these treated tumors. Marginal rims of viable tumor were occasionally noted in these lesions. histological heterogeneity was also found in the majority of the resected tumors from treatment groups IV (IL-2) and VI (RP + IL-2). The viable tumor cells at the periphery of the successfully
A 6-
I
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--g RPGY+ILP Fig 2. Survival. (A, B) Length of survival is noted after various combinations of preoperative immunotherapy were administered to mice with C1300-NB. Survival of all groups receiving IL-2 (IL-2, RF + IL-2, CY + IL-2, and RP + CY + k-2) was significantly prolonged compared with untreated controls fP < ,005). The longest survival was noted in the group treated with RP + CY + IL-2.
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VII (CY + IL-2). Tumors from CY + IL-2-treated animals measured 4.78 + 0.20 cm2 and were also significantly smaller than all groups (P < .OOl) except the RP + CY + IL-Ztreated mice (Figs 3A and 3B). Tumor Appearance and Histology
In the two treatment groups demonstrating the most profound effect on arresting tumor growth (RP + CY + IL-2 and CY + IL-2), all lesions were small and easily resectable at day 14 of therapy. In most of these animals, inflammatory process produced adherence of the tumor to the overlying sking. Otherwise, these tumors appeared encapsulated and did not grossly invade the underlying muscle. IL-2- and RP + IL-Ztreated mice survived until day 14 to undergo resection, but had extremely large tumors. None of these large tumors could be entirely extirpated and rapidly recurred after resection. Only a few mice from groups II (RP) and I (control) survived to undergo surgery. Their tumors were very large and not completely resectable.
“E 0
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Fig 3. Tumor growth rate. (A) single-agent therapies. No significant difference in tumor growth was observed over a ICday treatment period. (B) Tumor growth in all multiagent therapy groups was similar until day 5 of treatment. One day after IL-2 was begun [day 4). a divergence of growth rates was observed. At day 14, tumors from the group RP + CY + IL-2 were significantly smaller than those from the other groups (P < ,001). Lesions from CY + IL-2-treated mice were also statistically smaller than tumors from all groups except RP + CY + IL-2.
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s
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Fig 5. Class I expression of tumors from treatment groups as measured by mean fluorescence. A trend toward increased class I expression was noted in the groups that received IL-2. RP + CY + IL-2-treated animals exhibited statistically greater class I expression than all non-IL-2 groups and RP + IL-2-treated mice (P < .0035).
higher than the viable areas of resected tumor, which averaged 311.17 ? 24.07 (P = .0029) (Fig 6). Fig 4. Tumor appearance. C1300-NB tumors were resected at day 14 of treatment. Tumor seen at the top of the figure is from an untreated animal. Note the contrast between this large, friable hemorrhagic tumor and the small, fibrotic tumor below that was resected from a mouse treated with RP + CY + IL-2
treated nodules were no less pleomorphic or mitotitally active than those in the continually growing tumors. There were no significant lymphocytic infiltrates in any of the tumors. Tumors from group VIII (RP + CY + IL-2) appeared to be completely excised at operation. However 4 of 6 lesions recurred postoperatively and resulted in death of the animals within 13 to 22 days. Postoperative survival was significantly longer (P < .OOl) in this group than the other experimental groups in which recurrent tumors proved to be lethal in 4 to 16 days following surgery. Incomplete resections that left gross residual tumor resulted in tumorassociated deaths in 2 to 9 days. Class I Expression
As measured by mean fluorescence, there was a trend of higher class I expression in tumors from all groups treated with IL-2. Tumors from RP + CY + IL-2-treated mice measured 443.0 + 26.6, cm’, which was significantly higher than all other groups (P = .035), except groups VII (CY + IL-2; 400 2 47.4 cm’) (Fig 5). Histologically heterogeneous tumors exhibited both whitish necrotic and pink viable areas on cut section. These two separate areas were compared histologically in 7 tumors. The class I expression of the areas of coagulation necrosis measured 478.39 _+ 24.17 and was consistently and significantly
Vitamin A Levels
Serum vitamin A levels or normal, untreated A/J mice was 15 to 20 l.q$dL. In mice treated with RP, the serum level was elevated to a clinically nontoxic level of 25 to 35 Fg/dL. 600
I
P)
G 3 ii
100
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viable SECTION
OF
TUMOR
Fig 6. Class I expression from different areas of the same tumor; sections of the tumor that appeared whiie and fibrotic were compared with areas that appeared to be viable. Significantly increased class I expression was noted in the fibrotic areas (P < .OOzS).
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DISCUSSION The rationale for evaluating the three immunotherapeutic agents of low-dose CY, IL-2, and RP stems from previous studies. In a murine model of advanced C1300-NB, therapeutic responses were produced with surgical debulking of the tumor followed by immunomodulating doses of CY.’ The proposed mechanism of action of low-dose CY involves eradication of tumor-enhanced systemic T suppressor cell function. It was postulated that a CY-induced reduction in T suppressor activity might be synergistic with the effects of other immune modulators such as IL-2 and RP. Previous studies had determined that treatment with IL-2 alone was effective in improving survival of mice with IP C1300-NB.’ Retinoids were studied because of their demonstrated antineoplastic effects in a variety of other tumor models.12~‘5The mechanism of action of retinoids includes both tumoricidal and tumoristatic effects, immunomodulation, and cellular differentiation and maturation of NB cell lines.9*‘6Furthermore, synergistic antineoplastic effects of retinoids combined with CY,“-” and CY combined with IL-22@23 have been reported. In the present study, preoperative triple immunotherapy using CY, IL-2, and RP resulted in the best survival rate and the greatest inhibition of C1300-NB tumor growth when compared with all single- and double-drug regimens. A proposed mechanism by which this apparent synergism occurs is as follows. Previous studies demonstrated that the therapeutic effect of CY in the C1300-NB model involves eradication of tumor-enhanced T suppressor cell function.’ Moreover, it has been noted that CY-sensitive T suppressor cells can produce IL-2 inhibitors that neutralize the biological activity of IL-2.” Therefore, the observed synergism of CY and IL-2 may occur through CY-induced down-regulation of T suppressor cell function and reduction of IL-2 inhibitors, each resulting in elevated antitumor IL-2 activity. This may explain both the enhanced therapeutic effect of combined CY and IL-2 and the effectiveness of low doses of IL-2 when combined with CY therapy. The low dose of IL-2 used in the present study, 3.2 x lo5 III/d, was effective against C1300-NB and drug toxicity was limited. Toxicity-induced anorexia and lethargy were reversed by withholding therapy for 1 day after 6 days of therapy. As previously noted, the synergistic effects of CY combined with IL-2,?“-23 retinoids combined with CY,‘7-‘9and retinoids plus IL-2” have been studied. In combination these three immunotherapeutic agents may allow the IL-2 dose to be decreased even further. The use of nontoxic low doses of IL-2 constitutes a significant advantage over the high-dose IL-2 regimens currently being evalu-
305
ated in most human clinical trials. Severe capillary leak syndrome is a common complication of highdose IL-2 therapy, and unfortunately, has been accompanied by occasional mortality.” The addition of RP to CY + IL-2 therapy improved survival and further inhibited tumor growth. Vitamin A analogs have long been shown to demonstrate tumoricical and tumoristatic properties.‘“‘5 It is significant to note that their effectiveness in the treatment of NB has not yet been reported. The antitumor mechanism of retinoids is not well-defined. Immune potentiation by retinoids has been reported and includes increased accessory cell (macrophage and dendritic cells) functionn*% and increased Lyt-1 subset of mouse T lymphocyte population.‘2.29 Increased IL-2 production by spleen cells has also been observed in mice treated with vitamin A-acetate.z Direct cytotoxicity from retinoids may also occur from destabilization of cell membranes and release of lysosomal enzymes resulting in cell lysis.” Experimentally, additive antitumor effects have been noted when retinoid therapy is combined with radiation of chemotherapeutic agents, including CY.15.‘7-‘9 NB is similar to other malignant tumors of epithelial origin in that they exhibit low levels of class I MHC antigen (MHC I) on their cell surface.‘.’ Expression of MHC I antigen is a prerequisite for host cytolytic T cell (CTC) recognition and lysis of aberrant cells.’ The effectiveness of immunotherapy for NB may be limited by this phenomenon. If class I expression of NB could be upregulated, making tumors a better CTC target, immunotherapy may be more effective. Analysis of C1300-NB tumors for class I expression showed that the experimental group treated with RP + CY + IL-2 demonstrated the highest class I expression. Because this expression was significantly higher than that of untreated tumors, in vivo upregulation of class I expression appears to be possible. In fact, class I expression of all groups receiving IL-2 was significantly greater than the other experimental groups. Analysis of the viable and nonviable sections of treated tumors showed that class I expression was significantly increased in the areas that showed a pronounced therapeutic effect, as evidenced by the presence of coagulation necrosis. These conspicuous areas of coagulation necrosis weren noted only in the treatment groups that received IL-2. Up-regulated class I expression may have resulted from IL-2-induced autologous interferon activity as suggested by other studies.30 In experimental murine’ and human9 NB cell lines, class I expression has been increased by treatment with interferon. RP may have augmented these effects. It is an agent
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that has been shown to induce cellular antigenic modulation.9.‘6 and the morphological differentiation3”” of NB cells in culture. The RP + CY + IL-2 treatment group exhibited the best therapeutic response as well as the greatest class I expression. This observation reinforces the importance of immunotherapy-induced class I expression. In a study of histological specimens from children with NB, Squire et al noted the greatest class I expression in stage IV-S NB.’ IV-S NB is unique in that these lesions exhibit a remarkable tendency for spontaneous regression. This phenomenon may represent a type of host antitumor immune response similar to that observed in the present experimental model. In our current study, the duration of preoperative treatment was selected as 14 days because pilot studies demonstrated that the inhibitory effect on tujor growth was usually transient. RP + CY + IL-Ztreated tumors usually began increasing in size at 12 to 14 days of treatment. The resurgance of tumor growth both prior to or after resection may represent proliferation of resistance clones of cells within the lesion. Alternatively, tumor recurrence may be due to growth within areas that were not effectively treated because adequate doses of the therapeutic agents did not reach the lesion. Tumor regression induced by immunotherapy may be due to several factors. Whereas regression at the periphery of the tumor is probably dependent on host immunity, central hemorrhagic necrosis does not necessarily depend on immunologic factors.33 The similarity of the pattern of tumor necrosis induced by immunotherapy with RP + CY + IL-2 to that reported using other agents may be a clue to the mechanism of action. These histological changes are similar to those induced by interferon and tumor necrosis factor. In particular, the coagulation necrosis observed in the present study is very similar to the response produced by interferon as reported by Dvorak and Gresser.34 The pattern of tumor necrosis is also similar to that noted in mouse sarcomas treated by tumor necrosis factor, in which a viable rim
of tumor was noted peripheral to the area of central hemorrhagic necrosis.34 An interesting observation of this study was the fact that the admixture of irregular areas of viable tumor and necrosis were noted primarily within the RP + CY + IL-2- and CY + IL-2-treated tumors. One of the most significant concerns relating to the adequacy of therapy is the necessity of delivering therapeutic agents to the lesion in tumoricidal doses. Some areas within the tumor may be less vascular than normal tissue. Transport of chemotherapeutic agents into the tumor may be also reduced due to high intratumor extracellular fluid pressure. Circulation of antineoplastic agents within the tumor interstitium may also be limited by the large interstitial space and loss of fluid from the tumor’s periphery.35 In addition, the physical properties restricting delivery of traditional systemic chemotherapeutic agents probably depends on the size of the tumor. It may indeed be beneficial to reduce a large primary tumor into several smaller noncontiguous lesions separated by necrotic areas, as was observed in this study. The resulting residual nests of tumor tissue may possibly be more effectively treated with systemic agents. This study suggests that a combination of biological response modifiers administered at low nontoxic doses may be an effective component of multimode1 therapy. However, it is important to note that despite the initial antitumor response to combination immunotherapy, postoperative recurrences were frequent in all experimental groups. It appears that an modified regimen or additional postoperative therapy will be needed to improve survival. On the other hand, these agents seem to restore the balance of immunologic responsiveness in favor of the host. Most significant is the fact that these regimens might be used prior to operation without suppressing the patient’s immune defense or causing other serious complications commonly associated with standard chemotherapy or irradiation. Such well-tolerated therapeutic programs potentially could be used as maintenance therapy between pulses of standard chemotherapy following operation.
REFERENCES 1. Squire R, Fowler CL, Brooks SP, et al: The relationship of class I MHC antigen expression to stage IV-S disease and survival of neuroblastoma. J Pediatr Surg 25:381-386,199O 2. Sigal RK, Reynolds JV, Markmann JF, et al: Upregulation of MHC class I: Effect on growth and LAK sensitivity of neuroblastoma. Surg Forum 39:572-575,1988 3. Necheles TF, Tefft M, Weinberg V: Randomized trial of immunotherapy in the treatment of advanced neuroblastoma, in Terry WD, Rosenberg SA (eds): Immunotherapy of Human Cancer. New York, NY, Elsevier, 1982, pp 377-383
4. Bruce J, Brooks SP, Rich GA, et al: Effects of immunostimulation on host survival in A/J mice with transplantable Cl300 neuroblastoma. Curr Surg 45:17-19,1988 5. Fowler CL, Brooks SP, Rossman JE, et al: Postoperative immunotherapy of murine C1300-neuroblastoma. J Pediatr Surg 25:229-237, 1990 6. Brooks SP, Cooney DR, Fowler CL: Treatment of murine Cl300 neuroblastoma with IL-2/LAK cell therapy. Presented at the meeting of The Association for Academic Surgery, Salt Lake City, UT, November 16-19, 1988
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7. Tanaka K, Yoshioka T, Bieberich C, et al: Role of the major histocompatibility complex class I antigens in tumor growth and metastases. Annu Rev Immunol6:359-380, 1988 8. Lampson LA, Fisher LA, Whelan JP: Striking paucity of HLA-A. B, C and 82-microglobulin of human neuroblastoma cell lines. J Immunol130:2471-2478,1983 9. Gross N, Beck D, Favre S, et al: In vitro antigenic modulation of human neuroblastoma cells induced by IFN-gamma, retinoic acid and dibutyryl cyclic AMP. Int J Cancer 39:521-529,1987 10. Neeld JF Jr, Pearson WN: Macro- and micromethods for the determination of serum vitamin A using trifluoroacetic acid. J Nutr 79~454462, 1963 11. McCormick, DB: Vitamins, in Tietz NW (ed): Fundamentals of Clinical Chemistry. Philadelphia, PA, Saunders, 1986, pp 927-964 12. Eccles SA: Effects of retinoids on growth and dissemination of malignant tumors: Immunological considerations. Biochem Pharm 34:1599-1610,1985 13. Felix EL, Cohen MH, Loyd BC: Immune and toxic antitumor effects of systemic and intralesional vitamin A. J Surg Res 21:307-312, 1976 14. Watson RR, Moriguchi S, Gensler HL: Effects of dietary retinyl palmitate and selenium on tumoricidal capacity of macrophages in mice undergoing tumor promotion. Cancer Lett 36:181187.1987 15. Seifter E, Rettura G, Padawer J, et al: Regesssion of C3HBA mouse tumor due to x-ray therapy combined with supplemental B-carotene ofvitamin A. JNCI 71:409-417,1983 16. Reynolds CP, Maples J: Modulation of cell surface antigens accompanies morphologic differentiation of human neuroblastoma cell lines. Prog Clin Biol Res 17513-37, 1985 17. Nathanson L, Maddock CL, Hall TC: Exploratory studies of vitamin A and cyclophospbamide in tumor-bearing mice. J Clin Pharm 9:359-373,1969 18. Levenson SM, Rettura G, Seifter E: Effects of supplemental dietary vitamin A and B-carotene on experimental tumors, local tumor excision, chemotherapy, radiation injury, and radiotherapy, in Butterworth CE Jr, Hutchinson ML (eds): Nutritional Factors in the Induction and Maintenance of Malignancy. New York, NY, Academic, 1983, pp 169-203 19. Cohen MH, Carbone PP: Enhancement of the antitumor effects of 1,3-bis (2-chloroethyl)-1-nitrosourea and cyclophosphamide by vitamin A. JNCI 48:921-926,1972 20. Lee K, O’Donnell RW, Marquis D, et al: Eradication of palpable intradermal murine bladder tumors by systemic interleukin-2 and cyclophosphamide in C3H mice. J Biol Response Mod 7:32-42,1988 21. Hosokawa M, Yabiku T, Ikeda J, et al: Effects of a combination of cyclophosphamide and human recombinant inter-
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Discussion G. Haase (Denver, CO): The authors have presented an elegant laboratory study evaluating preoperative immunotherapy in the murine Cl300 neuroblastoma model. It is a reasonable postulate that these three agents in combination could inhibit tumor growth and improve survival. Although the classic experimental model used is immunogenic, its use comprises the study. Why was one of several well-
established human neuroblastoma cell lines not used? The impact of the biological response modifiers might then have been more easily transferred to the clinical setting. I am also concerned that recombinant human IL-2 was used. What effect does this human product, which may function by a different mechanism, have on a murine cell line? The biggest impact on tumor
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growth and survival occurred in experimental groups receiving the IL-2, so the validity of these observations is under question. The authors conclude that a combination of these agents administered at low doses would be part of an effective human therapeutic regimen. This is contrary to current oncologic thought (the Coldie-Goldman hypothesis) that antitumor therapy should be given intensely, up-front, and in the highest possible does consistent with cell cycle kinetics followed by a rescue technique such as bone marrow transplantation. With this current clinical emphasis on dose intensity, how important do the authors think their experimental results really are? C. Fowler (response): Dr. Haase, the reason we used the murine model is that we have had experience with it for several years. The reason we don’t at present evaluate immunotherapy on human cell lines is that although there are numerous human cell lines available, we were trying to establish an in vivo model because obviously the effects of immunotherapy will be different in culture and in vivo. Human interleukin was used because it is readily available and immunologically, as far as we know, it acts the same as a mouse interleukin. The low doses we used are contrary to what the oncologists use. Oncologists teach that “more is better,” but when you see patients with numerous toxic side effects, it is apparent that lowdose immunotherapy should provide some advantage over high-dose chemotherapy because the low doses we were using were nontoxic. Using nontoxic low doses to enhance the patient’s own host immune defense should be better than completely immunocompromising a patient with high-dose therapy and then rescuing him/her with other drugs. This is especially the case in neuroblastomas, because standard chemotherapy has not been shown to satisfactorily improve the prognosis of children with advanced disease. R.M. Filler (Toronto, Ontario): Could you perhaps theorize why this works that way? Is there any indication that the resistance of the cells to chemotherapy is changed? C. Fowler (response): In this paper, we haven’t gone into the mechanisms of action. Earlier, we showed in this model that low-dose cyclophosphamide in the
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doses that we use presently decreases an increased T-suppressor cell activity that Cl300 neuroblastoma induces. Vitamin A in the literature has been shown to be tumorcidal and tumoristatic. It has increased accessory cell function and what is especially interesting in pertaining to neuroblastoma is that if you put any one of the retinoids in culture with either human or mouse neuroblastoma, it will produce a differentiation and maturation of the cells. That’s only been seen in vitro not in vivo, but possibly in our in vivo Cl300 neuroblastoma model, retinoids could enhance some of the other immunotherapies that we are using to produce this same effect. IL-2 is a lymphokine that activates the other T cells. Other papers have been written about synergism between vitamin A and IL-2 as well as vitamin A and cyclophosphamide. Vitamin A increases cell membrane labilization so that agents such as cyclophosphamide can enter the tumor cell more easily and produce their effects. Qclophosphamide and IL-2 also have been shown to be synergistic. Cyclophosphamide decreases IL-2 inhibitors produced by T suppressor cells, thus enhancing IL-2 effects. The histological changes that we’ve seen on cut specimens of treated tumors included coagulation necrosis and rims of viable tumor. In the literature interferon produces some of these effects, so perhaps interferon was induced as part of the immunotherapy. This is further supported in that interferon is known to increase class I expression and increased class I expression was also noted in the treated tumors. Finally, tumor necrosis factor is another possible mechanism. It produces hemorrhagic necrosis and a viable rim of tumor, a pattern that we have seen in some of these tumors. Our next experiments will be to see exactly which mechanisms are used in among all these possibilities. D. Tapper (Seattle, WA): Dr Fowler, can I ask you two things. Is your neuroblastoma model a nonmetastasizing logo tumor model? Is that correct? The purpose then, unlike the other IL-2 studies, is to measure logo changes in the tumor? To go back to what Dr Haase asked, do you know the genetic make-up of this tumor? C. Fowler (response): The clone we have is nonmetastasizing. No.
