Adjuvant Therapy for Melanoma in Dogs

Vol. 5, 4249–4258, December 1999 Clinical Cancer Research 4249

Adjuvant Therapy for Melanoma in Dogs: Results of Randomized Clinical Trials Using Surgery, Liposome-encapsulated Muramyl Tripeptide, and Granulocyte Macrophage Colony-stimulating Factor1

E. Gregory MacEwen,2 Ilene D. Kurzman, colony-stimulating factor (rcGM-CSF) in dogs undergoing David M. Vail, Richard R. Dubielzig, surgery for oral melanoma. Ninety-eight dogs with histologically confirmed, clini- Karen Everlith, Bruce R. Madewell, cally staged, oral melanoma were entered into two random- 3 Carlos O. Rodriguez, Jr., Brenda Phillips, ized, double-blind, surgical adjuvant trials. In trial 1, 50 Courtney H. Zwahlen,4 Joyce Obradovich, dogs were stratified based on clinical stage and randomized Robert C. Rosenthal, Leslie E. Fox, to once a week L-MTP-PE or lipid equivalent (control). Mona Rosenberg, Carolyn Henry, and When all of the clinical stages were combined, no difference 5 in disease-free survival or in survival time (ST) were de- Janean Fidel tected. However, within stage I, dogs receiving L-MTP-PE University of Wisconsin, School of Veterinary Medicine, Madison, had a significant increase in ST compared with control, with Wisconsin 53706 [E. G. M., I. D. K., D. M. V., R. R. D., K. E., B. P.]; 80% of the dogs treated with L-MTP-PE still alive at >2 University of California, School of Veterinary Medicine, Davis, years. Within each stage II and stage III, there was no California 95616 [B. R. M., C. O. R., B. P., C. H. Z.]; Oakland Veterinary Referral Services, Bloomfield Hills, Michigan 48302 difference detected between the treatment groups. In trial 2, [J. O.]; Veterinary Specialists of Rochester, Rochester, New York 48 dogs were stratified on the basis of clinical stage and 14623 [R. C. R.]; University of Florida, College of Veterinary extent of surgery (simple resection or radical excision), Medicine, Gainesville, Florida 32610 [L. E. F.]; Veterinary Cancer treated with L-MTP-PE two times a week, and randomized Referral Group, Los Angeles, California 90064 [M. R.]; University of to rcGM-CSF or saline (placebo) given s.c. daily for 9 weeks. Missouri, College of Veterinary Medicine, Columbia, Missouri 65211 Within each stage and when all of the stages were combined, [C. H.]; and Special Veterinary Services, Berkeley, California 94704 [J. F.] there was no difference between the treatment groups. In both studies, stage I COM is associated with a better prognosis. No effect on survival was observed with regard to ABSTRACT tumor location in the oral cavity, sex, type/extent of surgery, Spontaneous canine oral melanoma (COM) is a highly or age. In a subset of dogs tested, pulmonary alveolar metastatic cancer, resistant to chemotherapy, and can serve macrophage cytotoxicity was enhanced with combined as a model for cancer immunotherapy. Liposome-encap- rcGM-CSF and L-MTP-PE but not in dogs treated with sulated muramyl tripeptide-phosphatidylethanolamine (L- L-MTP-PE alone. MTP-PE) can activate the tumoricidal activity of the mono- The present study indicates that after surgery, cyte-macrophage system following i.v. injection. The L-MTP-PE administered alone or combined with rcGM- objective of these studies was to evaluate the therapeutic CSF showed no significant antitumor activity in treating effectiveness of L-MTP-PE administered alone and com- advanced stage COM. In early stage COM, L-MTP-PE was bined with recombinant canine granulocyte macrophage shown to result in a prolongation of ST. Furthermore, this study provides additional rationale for the use of the dog model for human malignant melanoma.

INTRODUCTION Received 3/4/99; revised 8/30/99; accepted 9/21/99. There is ample evidence that the immune system can The costs of publication of this article were defrayed in part by the modulate the progression and metastasis of malignant mela- payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to noma. Several biological agents and approaches have demon- indicate this fact. strated antitumor activity (1–10). The most widely used treat- 1 This work was supported by a grant from the Morris Animal Foun- ments include IFN-␣ (5, 6), IL-26 and adoptive immunotherapy dation, Englewood, CO, and Ciba-Geigy Ltd., Basel, Switzerland. 2 To whom requests for reprints should be addressed, at Department of Medical Sciences, University of Wisconsin, 2015 Linden Drive West, Madison, WI 53706. 3 Present address: University of Texas, M. D. Anderson Cancer Center, 6 The abbreviations used are: IL, interleukin; DFS, disease-free survival; 1515 Holcombe Boulevard., Houston, TX 77054. ST, survival time; PAM, pulmonary alveolar macrophage; MTP-PE, 4 Present address: Southwest Surgery and Oncology, 17672 Cowan muramyl tripeptide-phosphatidylethanolamine; L-MTP-PE, liposome- Avenue, Irvine, CA 92614. encapsulated MTP-PE; MLV, multilamellar vesicle; OSA, osteosar- 5 Present address: University of Zurich, Winterthurerstrasse 260, CH- coma; GM-CSF, granulocyte macrophage colony-stimulating factor; 8057 Zurich, Switzerland. rcGM-CSF, recombinant canine GM-CSF; ADCC, antibody-dependent

free of disease at 24 weeks (33). In a follow-up report on these same 18 patients, 4 remain free of disease more than 5 years after surgical resection and L-MTP-PE therapy (40). Undoubt- edly, the greatest antitumor activity of L-MTP-PE has been demonstrated in children with metastatic OSA leading to a Phase III randomized trial presently underway (41, 42). Our group has focused on spontaneous canine tumors as models for immunotherapy (43). We have previously reported the antimetastatic activity of L-MTP-PE, administered alone or in combination with chemotherapy, in surgical adjuvant randomized clinical trials in dogs with spontaneous OSA (44, 45) and hemangiosarcoma (46). In over 125 dogs with OSA, L- MTP-PE was found to prolong metastasis-free and overall STs when given alone or after systemic chemotherapy. In dogs with Fig. 1 Schematic diagram of the two oral melanoma clinical trials. hemangiosarcoma, L-MTP-PE administered concurrently with doxorubicin and cyclophosphamide, resulted in prolonged metastasis-free and overall STs compared with dogs receiving chemotherapy alone (46). (11), and active immunization using tumor vaccines (12–15). GM-CSF is a cytokine with a unique ability to stimulate Advances in melanoma treatment over the past decade have differentiation of hematopoietic progenitor cells into dendritic been directed at the role of adjuvant systemic therapy for pa- cells, a potent antigen-presenting cell (47, 48). GM-CSF has tients with melanoma at high risk of recurrence or metastasis (1, received particular attention for enhancing the antitumor activity 9, 10, 16). in transduced tumor cell vaccines (14, 15, 49). In vitro, GM- Canine melanoma is a highly metastatic spontaneous tumor CSF has been shown to slightly enhance cytotoxic activity of arising most commonly in the oral cavity (17–19). COM has a peripheral blood monocytes and lymphocytes and to markedly biological behavior that is similar to oral melanoma in humans. increase ADCC (50, 51) and enhanced monocyte cytotoxicity Melanomas are the most common neoplasm seen in the oral against a melanoma cell line (52). In vivo administration of cavity in dogs (20–22). These tumors arise in many breeds but GM-CSF in human cancer patients enhanced monocyte ADCC are more frequently identified in the dark pigmented breeds (53) and increased secretion of both TNF-␣ and IFN (54). In (e.g., Scottish terrier, Boston terrier, Airedale, and Cocker span- another study in cancer patients, GM-CSF enhanced monocyte iel). COM occurs most often in dogs that are 8–12 years of age, cytoxicity against colon HT29 tumor cells, although no antitu- roughly corresponding to the ages of highest prevalence in mor responses were seen (55). humans (40–60 years; Ref. 22). Metastasis is common and can This study was designed in a randomized manner to be seen in regional lymph nodes, lung, liver, brain, and kidney. evaluate the efficacy of L-MTP-PE when used in a surgical The treatment of choice is surgical excision, and, depending on adjuvant setting and administered alone or in combination with clinical stage, the median ST is 8–10 months, with death due to GM-CSF. metastasis (18, 19, 23, 24). The systemic activation of macrophages using a variety of techniques has been shown to inhibit the proliferation of or to MATERIALS AND METHODS destroy a wide range of syngeneic, allogeneic, and xenogeneic Patient Population. Ninety-eight dogs with previously tumors (25, 26). In rodents, dogs, and humans, a liposome- untreated, histologically confirmed spontaneous oral melanoma, encapsulated lipophilic derivative of muramyl dipeptide, known without radiographic evidence of distant metastasis, were stud- as MTP-PE, when administered in vivo can activate monocytes ied. The pretreatment evaluation included a complete physical and macrophages resulting in antitumor activity (27–38). It has examination, three-dimensional measurements of the primary been shown that L-MTP-PE can result in the regression of tumor, a complete blood count, serum chemistry profile, and established metastasis in the B16 murine melanoma model (39). urinalysis. For evaluation of metastatic disease, regional lymph In humans with melanoma, two Phase II trials using L-MTP-PE nodes were evaluated with fine needle aspiration or biopsy and have been reported (32, 33). In one study, 30 patients with radiographs of the thorax. Only dogs without evidence of overt measurable metastatic melanoma were treated, and, although no lung metastasis, in overall good health, and a tumor (including responses were detected, peripheral blood monocyte cytotoxic- regional lymph nodes) amenable to complete surgical removal ity was increased (32). In the other study, 18 stage III melanoma were eligible for entry into this study. Depending on the location patients with resectable tumors were treated with surgery and of the primary tumor, radiographs of the mandible or maxilla L-MTP-PE. After the administration of L-MTP-PE, enhanced were performed under general anesthesia to determine the extent monocyte cytotoxicity was detected in 6 of the 9 patients still of local invasion and to assist in surgical planning. Written consent for entry into these trials was obtained from each dog’s owner before entry into this study. Treatment. Primary treatment was surgical removal of cellular cytotoxicity; TNF, tumor necrosis factor; COM, canine oral the primary tumor (stage I and II) and regional lymph nodes melanoma; BAL, bronchoalveolar lavage. (stage III). For tumors involving the gingival region—

Table 1 Patient characteristics Trial 1 Trial 2 L-MTP-PE ϩ L-MTP-PE ϩ Parameter L-MTP-PE MLV rcGM-CSF saline Age (yr)a 11.8 (11.6); 4.5–20 10 (10.2); 0.7–16 9.5 (9.7); 5–16 8.5 (9.2); 5–14 Weight (kg)a 18 (21.5); 5–43.7 24.7 (23.5); 3.7–47 22 (24.4); 1.9–53 26.8 (28.4); 5.8–50 Clinical stage, n I 11 9 13 12 II81178 III 6 5 4 4 Sex, n Male-intact 5 5 2 6 Male-neutered 10 9 11 11 Female-intact 2 2 0 0 Female-spayed 8 9 11 7 a Median (mean); range.

especially if bone involvement was detected in the mandible or Geigy, Ltd. (Basel, Switzerland). Liposomes were prepared maxilla—a partial or complete mandibulectomy or partial max- from a freeze-dried preparation by the addition of buffered illectomy was performed as described previously (56, 57). The saline, without calcium or magnesium, to vials containing dio- decision to perform this procedure was at the discretion of the leoyl-phosphatidylserine and 1-palmitoyl-2-oleoyl-phosphatid- surgeon. All of the dogs entered were considered free of clini- lycholine at a 3:7 molar ratio, with (L-MTP-PE) or without cally detectable tumor after surgery. After surgery, all of the (MLV) MTP-PE. The ratio of active ingredient (MTP-PE) to dogs were clinically staged, according to the WHO staging phospholipid was 1:250. After 1 min, the vial contents were system, into stage I (tumors Ͻ2-cm diameter, node negative), agitated on a vortex mixer or vigorously shaken by hand for 1 stage II (2- to 5-cm diameter, node negative), or stage III min, and then the contents were diluted with buffered saline to (Ͼ5-cm diameter and/or node positive). This was a multicenter a concentration of 25 mg lipid/ml. trial in which dogs from nine veterinary facilities were entered. GM-CSF. rcGM-CSF was kindly provided by Amgen, One investigator (I. D. K.) performed all of the randomization. Inc. (Thousand Oaks, CA). Dogs were initially administered 15 Drugs for each dog entered were prepared at the University of ␮g/kg s.c. daily for 9 weeks, a dose that we found to enhance Wisconsin-Madison and sent by overnight express mail to the monocyte tumor cytostasis (58) and is well tolerated in normal participating investigator. A schematic diagram of the protocols experimental dogs. Dogs were started on rcGM-CSF or saline for the two trials is shown in Fig. 1. for 1 week prior to L-MTP-PE treatment and continued concur- Trial 1. After surgery and stratification based on clinical rently with L-MTP-PE for 8 weeks. The rationale to begin stage, dogs were treated in a double-blind fashion with L- rcGM-CSF 1 week before L-MTP-PE was to allow for an MTP-PE or lipid equivalent (MLV) once a week for 8 weeks. increase in absolute monocyte count before the administration The liposomes were given via a slow i.v. infusion over 5–8 min. of L-MTP-PE. Because of toxicity in 10 dogs in the early phase The first dose of L-MTP-PE was 1 mg/m2; all of the subsequent of trial 2, the dose of rcGM-CSF was reduced to 5 ␮g/kg/day. doses were scaled according to the weight of the dog (1 mg/m2 Follow-Up Evaluation. In both trials, dogs were evalu- for dogs Ͻ5 kg, 1.5 mg/m2 for dogs 5–10 kg, and 2 mg/m2 for ated with routine physical examination and thoracic radiographs dogs Ͼ10 kg). This dose of L-MTP-PE was similar to that used at 3-month intervals. Physical and historical abnormalities were in previous studies (41–43). investigated as clinically indicated at each recheck visit. Patient Trial 2. After surgery, dogs were stratified by clinical evaluations continued as long as necessary to determine DFS stage. The extent of surgery was left to the discretion of the and overall ST for each dog. The DFS was defined as the time surgeon. To minimize potential bias related to the extent of from surgery to evidence of clinical recurrence or metastasis. ST surgery, dogs were further stratified on the basis of type/extent was defined as the time from surgery to death or euthanasia due of surgery. Surgery was either radical excision (mandibulec- to advanced disease. Euthanasia was performed when requested tomy or maxillectomy) versus simple excision (complete exci- by the dog’s owner because the animal’s quality of life was sion of tumor but not involving removal of bone). After strati- considered severely limited due to advanced disease. fication, the dogs were randomized to receive rcGM-CSF (initial Cytotoxicity Assays. A subset of dogs in trial 2 was dose 15 ␮g/kg) or saline, s.c. daily for 9 weeks. The frequency tested to determine PAM cytotoxic activity after L-MTP-PE of administration of L-MTP-PE was increased from once a week with or without rcGM-CSF. PAM cytotoxic activity was (in trial 1) to two times a week using the same dosage as evaluated before rcGM-CSF or saline was administered, im- described for trial 1. L-MTP-PE was increased from once a mediately prior to the first L-MTP-PE treatment and two days week to two times a week because of the lack of therapeutic after the second L-MTP-PE treatment. Dogs were placed efficacy detected in stage II and stage III dogs in trial 1. under general anesthesia and PAMs were collected via BAL, Liposome Preparation. Lyophilized liposomes with or using a cold-sterilized fiberoptic bronchoscope and sterile without MTP-PE (CGP 19835A lipid) were provided by Ciba- saline according to the procedure described previously (59).

Table 2 Analysis of DFS and overall STs No. of Median DFS Median ST dogs (days) P (days) P Trial 1: L-MTP-PE or MLV All stages combined L-MTP-PE 25 346 0.353 504 0.332 MLV 25 174 271 Stage I L-MTP-PE 11 a 0.220 a 0.045 MLV 9 396 414 Stage II L-MTP-PE 8 152 1.000 254 0.943 MLV 11 150 293 Stage III L-MTP-PE 6 85 0.779 338 0.322 MLV 5 147 157 Stage II ϩ III L-MTP-PE 14 152 0.966 258 0.925 MLV 16 156 243 Treatment groups combined Stage I 20 477 0.020 a 0.015 Stage II 19 154 257 Stage III 11 147 226 Trial 2: L-MTP-PE or rcGM-CSF All stages combined rcGM-CSF 24 212 0.545 498 0.892 Saline 24 290 501 Stage I rcGM-CSF 13 489 0.480 573 0.230 Saline 12 532 a Stage II rcGM-CSF 7 118 0.846 261 0.940 Saline 8 117 228 Stage III rcGM-CSF 4 35 0.654 a 1.000 Saline 4 92 275 Stage II ϩ III rcGM-CSF 11 90 0.948 286 0.840 Saline 12 112 306 Treatment groups combined Stage I 25 622 0.0003 653 0.049 Stage II 15 118 248 Stage III 8 48 364 Trial 1 ϩ Trial 2 Frequency of L-MTP-PE L-MTP-PE 1/wk 25 346 0.837 504 0.917 L-MTP-PE 2/wk 48 275 539 Effect of surgery Simple resection 62 276 0.645 584 0.397 Radical surgery 36 237 460 a Median not yet reached.

PAMs were isolated from lavage fluid using sterile, endotox- herence, nonadherent PAMs were gently removed. To deter- in-free density gradient separation (Ficoll-Hypaque, specific mine purity of the adherent cell population, cytospin prepa- gravity 1.077, Sigma Chemical Company, St. Louis, MO). rations (Cytospin-2 Shandon Inc. Pittsburgh, PA) were made The coculture cytotoxicity assay used has been recently de- of the cells. The cytospin preparations were stained with scribed in dogs (60). Briefly, PAMs were added to wells of a Diff-Quick (American Scientific Products, McGaw Park, IL) 96-well microtiter plate and allowed to adhere for 90 min at and examined microscopically. After a 24-h incubation, 3H-

37°C in 5% CO2 humidified atmosphere. PAMs were cul- labeled CML-6M-C2 canine melanoma cells (kindly pro- tured in RPMI 1640 (Life Technologies, Inc., Grand Island, vided by L. Wolfe, Auburn University, Auburn, AL) were NY) supplemented with 10% heat-inactivated FCS (Intergen, added to each well in E:T ratios of 2.5:1, 5:1, and 10:1. Purchase, NY), and 2 mM sodium pyruvate, 2 mML-gluta- Tumor cells were added to additional wells for the determi- 3 mine, 10 mM HEPES buffer, 100 units/ml penicillin, and 100 nation of spontaneous and maximal release of H. Cells were ␮g/ml streptomycin (Sigma Chemical Company). After ad- cocultured for 72 h, and then, the radioactivity (cpm) of 50 ␮l

Fig. 2 DFS (A) and ST (B) for dogs in trial 1 treated once weekly with L-MTP-PE or MLV. There was no difference in survival between the treatment groups.

Fig. 3 DFS (A) and ST (B) for dogs in trial 1 with stage I oral melanoma, treated once weekly with L-MTP-PE or MLV. Dogs that received L-MTP-PE had significantly prolonged ST compared with dogs that received MLV; P Ͻ 0.045. There was no significant difference between treatment groups with regard to DFS.

of supernatant from each well was determined. Percent cy- stages combined) is 8 months (18, 23); we anticipated a 40% totoxicity of CML-6M-C2 cells was calculated by the fol- increase in the median survival in dogs treated with L-MTP-PE. lowing formula: With 25 dogs per arm, we should be able to detect that difference with an 80% power at the P ϭ 0.05 level. For trial 2, we expected T Ϫ S ϫ a median ST of 11 months for surgery and L-MTP-PE, and we Ϫ 100 M S anticipated a 40% increase in the median survival with the addition where T ϭ mean cpm of supernatant from wells containing of rcGM-CSF. With 25 dogs per arm, we would be able to detect ϭ tumor and effector cells, S (spontaneous release) ϭ mean cpm of that difference with an 80% power at the P 0.05 level. supernatant from wells containing tumor cells alone, and M DFS and overall ST were compared between the treatment (maximal release) ϭ mean cpm of lysate from wells containing groups of both trials. STs were also compared between dogs tumor cells alone. receiving L-MTP-PE once a week in trial 1 and dogs receiving Anesthesia for the BAL. Dogs were premedicated with L-MTP-PE two times a week in trial 2. Survival curves were midazolam (0.2 mg/kg, maximum of 5 mg, s.c.) and butorphanol generated by the Kaplan-Meier method and were compared (0.2 mg/kg, maximum of 10 mg, s.c.) 30 min before anesthesia. using the Breslow and Mantel-Cox tests of significance between Anesthesia was induced by injectable thiopentothal sodium (12–15 survival curves (61–63). These statistical tests adjust for dogs mg/kg) and maintained on isoflurane and 100% oxygen, using an still alive or lost to follow-up at the time of analysis. A P of less endotracheal tube. A bronchoscope was passed through the larynx, than 0.05 was considered significant. and the BAL was performed as described previously (59). Differences in percent cytoxicity were assessed using Statistical Analysis. In designing trial 1 in dogs with oral paired t tests. For results covering a range of E:T ratios, the melanoma, the expected median ST for surgery alone (all of the highest P is reported.

Fig. 4 DFS (A) and ST (B) for dogs in trial 2 when all of the stages were combined and the dogs were grouped by treatment (L-MTP-PE two times a week combined with either daily rcGM-CSF or daily saline). No differences in survival were observed with regard to treatment.

Fig. 5 DFS (A) and ST (B) for all of the dogs that received L-MTP-PE (trials 1 and 2 combined) once a week or two times a week. No differences in survival were observed with regard to the frequency of administration of L-MTP-PE.

RESULTS between dogs treated with L-MTP-PE versus placebo (Fig. 2). Patient Characteristics. Patient characteristics for all of However, within stage I, dogs receiving L-MTP-PE had signif- the dogs entered into both trials are given in Table 1. icantly prolonged ST (P Ͻ 0.05) compared with those receiving Treatment and Survival Data. The L-MTP-PE treat- MLV (Fig. 3). In the L-MTP-PE group, 80% of the dogs were ments were well tolerated by the dogs. The only consistent side alive at 2 years compared with 25% in the MLV group. The effect was an elevation in body temperature (1–2°C), lasting statistical difference detected is based on 11 dogs treated with 1–4 h after the liposome treatment. The temperature elevation L-MTP-PE and 9 dogs treated with MLV (control). Increasing was more pronounced in the L-MTP-PE groups. Body temper- the patient population with stage I COM would increase the ature returned to normal within 6 h after injection. The fever power of this observation. However, the 25% 2-year ST for the response was most apparent on the first few treatments and then stage I dogs treated with MLV is similar to previous ST for dogs subsided on subsequent treatments. In trial 2, the initial dosage treated by surgery alone (23). for rcGM-CSF was 15 ␮g/kg s.c. daily. However, 10 dogs When treatment groups were combined, stage I (n ϭ 20) experienced adverse side effects that included thrombocytope- had significantly prolonged (P Ͻ 0.022) DFS and ST compared nia (n ϭ 4), anterior uveitis (n ϭ 2), lethargy and mild diarrhea with stage II (n ϭ 19) and stage III (n ϭ 11). The prognostic (n ϭ 2), gastritis (n ϭ 1), and polydipsia/polyuria (n ϭ 1). After significance of the clinical staging system confirms similar documentation of these adverse side effects, all of the subse- findings in a previously published study (23). No effect on DFS quent dogs were treated at a dose of 5 ␮g/kg. Table 2 summa- or ST was observed with regard to the location of the primary rizes the median DFS and ST for dogs according to clinical tumor, type of surgery, sex, or age (data not shown). stage and treatment group for both trial 1 and 2. Trial 2. Forty-eight dogs were entered into this clinical Trial 1. Fifty dogs were entered into this clinical trial. trial. All of the dogs received L-MTP-PE two time a week, and Twenty-five were randomized to L-MTP-PE and 25 to MLV. 24 were randomized to receive rcGM-CSF and 24 to receive When all of the stages were combined, there was no difference saline. Within each stage and with all of the stages combined,

Fig. 6 Cytotoxic activity against CML-10 canine melanoma cells by PAM from five dogs in trial 2 that received L-MTP-PE combined with either rcGM-CSF (A, n ϭ 3) or saline (B, n ϭ 2). PAM cytotoxic activity was assessed before treatment (Pretreatment), at day 7 of rcGM-CSF or saline treatment (before L-MTP-PE), and two days after the second L-MTP-PE treatment (Post L-MTP-PE). Cytotoxic activity was significantly enhanced by L-MTP-PE in the dogs that received rcGM-CSF. This effect was not observed in the dogs that received saline.

there was no difference in DFS or ST when dogs receiving survival advantage (4, 66, 67). One study using C. parvum showed L-MTP-PE and rcGM-CSF were compared with dogs receiving a survival advantage in a surgical adjuvant trial in COM (23). No L-MTP-PE and saline (Fig. 4). No effect on DFS or ST was randomized clinical trials have been conducted using adjuvant observed with regard to the location of primary tumor, type of L-MTP-PE in human melanoma. The trials presented in this paper surgery, sex, or age (data not shown). are the first randomized surgical adjuvant trials using L-MTP-PE in Trials 1 and 2 Combined. Comparison of dogs receiv- spontaneous malignant melanoma. ing L-MTP-PE once a week (trial 1) or two times a week (trial Patient characteristics for the population studied were similar 2) showed no difference in DFS or ST with regard to frequency to those reported elsewhere (17–19, 23, 68). The results of these of L-MTP-PE (Fig. 5). Further analysis on type of surgery trials show that L-MTP-PE, when administered in a surgical adju- (simple versus radical), revealed no significant difference in vant setting, has minimal antitumor activity, as evidenced in dogs DFS or ST (Table 2). with early stage (stage I) COM. Our study demonstrated a signif- Cytotoxicity Assays. In 5 dogs, PAMs were collected icant reduction in metastasis as evidenced by an increase in ST, by BAL and were assayed for cytotoxic activity against the with 80% having long-term survival (Ͼ2 years). However, this canine melanoma cell line, CML-6M-C2, before and after difference was seen only in stage I, and the number of dogs ϭ ϭ rcGM-CSF (n 3) or saline (n 2) and L-MTP-PE. Mean randomized within this stage was small (n ϭ 20). Although only 9 Ϯ ( SE) levels for percent cytotoxicity are shown in Fig. 6. dogs were treated with MLV (surgery alone), our previous studies ϭ Cytotoxic activity was markedly increased (P 0.053) in the have shown that stage I dogs with COM that are treated with rcGM-CSF group after receiving L-MTP-PE. At a 10:1 Et surgery alone have a similar median ST (23). In trial 2, all of the ratio for PAMs:tumor target, the pretreatment cytotoxicity dogs were treated with L-MTP-PE, and the median ST for stage I against CML-6M-C2 was a mean of 13% and increased to dogs was 622 days, which suggests a greater therapeutic efficacy 68% after rcGM-CSF and L-MTP-PE administration. In the than other studies using surgery alone (18, 23). group of dogs receiving L-MTP-PE plus saline, no increase in An important change made between trial 1 and trial 2 was in PAM cytotoxicity was detected (Fig. 6). increasing the administration of L-MTP-PE from once a week to two times a week. This change was made to determine whether DISCUSSION two-times-a-week treatment would increase the therapeutic re- The present surgical adjuvant therapy for stages I, II, and III sponse in stage II and III dogs. Results from trial 2 demonstrated (American Joint Committee on Cancer) melanoma is the use of that increasing the treatment frequency to two times a week did not high-dose IFN-␣ (10). Before the approval of IFN-␣, a large enhance the therapeutic activity of L-MTP-PE. In previous studies number of randomized trials evaluated the use of nonspecific using rodent models, two-times-a-week treatments were selected bacteria immunostimulants in the surgical adjuvant setting. Bacille because L-MTP-PE was shown to activate macrophage tumoricidal Calmette-Guerin, the bovine mycobacterium used as a tuberculosis function for 48–72 h, and two-times-a-week treatment would vaccine, has been extensively tested (2, 64, 65). A total of 13 maintain that activation (25, 69). In comparing the results of trials published trials involving 1152 patients have been reported (10). 1 and 2, no difference between once- or twice-weekly L-MTP-PE None of these randomized controlled studies has shown any sig- administration was seen in dogs with stage II or stage III disease. nificant impact on relapse-free or overall survival in the treated Another interesting finding from this study was the dem- patients. A heat-killed preparation of Corynebacterium parvum onstration that the extent of surgery (simple resection versus (Propionobacterium acnes) has been shown to provide a slight radical resection) had no significant influence on DFS and ST survival advantage (3); however, other trials did not show any (Table 2). Other reported studies in COM using radical surgery

alone (maxillectomy and mandibulectomy) report median STs tered in a surgical adjuvant setting. However, there is suggestive of 4.5–10 months (56, 57, 70–72). These STs compare favor- evidence that L-MTP-PE results in the prolongation of survival ably with the median STs reported in this study. This indicates in dogs with early (stage I) malignant COM. Furthermore, this that the major concern with COM is metastasis, and therapy study helps to strengthen the rationale for the use of the dog as must be directed toward metastasis control. a model for human spontaneous malignant melanoma. We did demonstrate that systemic administration of L-MTP-PE in dogs after the administration of rcGM-CSF will activate the tumoricidal properties of PAM in dogs with melanoma. ACKNOWLEDGMENTS We have previously reported that PAMs, collected using BAL, can We thank W. C. Kisseberth, C. A. London, L. Kravis, R. Chun, D. be activated in vitro using IFN-␥ (60, 73). We have found that the Thamm, E. 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E. Gregory MacEwen, Ilene D. Kurzman, David M. Vail, et al.

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