Clinical Cancer Research The Science of Cancer Health Disparities
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by MacEwen, E. G.
Right arrow Articles by Fidel, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by MacEwen, E. G.
Right arrow Articles by Fidel, J.
Clinical Cancer Research Vol. 5, 4249-4258, December 1999
© 1999 American Association for Cancer Research

Experimental Therapeutics, Preclinical Pharmacology

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 MacEwen2, Ilene D. Kurzman, David M. Vail, Richard R. Dubielzig, Karen Everlith, Bruce R. Madewell, Carlos O. Rodriguez, Jr.3, Brenda Phillips, Courtney H. Zwahlen4, Joyce Obradovich, Robert C. Rosenthal, Leslie E. Fox, Mona Rosenberg, Carolyn Henry and Janean Fidel5

University of Wisconsin, School of Veterinary Medicine, Madison, Wisconsin 53706 [E. G. M., I. D. K., D. M. V., R. R. D., K. E., B. P.]; University of California, School of Veterinary Medicine, Davis, California 95616 [B. R. M., C. O. R., B. P., C. H. Z.]; Oakland Veterinary Referral Services, Bloomfield Hills, Michigan 48302 [J. O.]; Veterinary Specialists of Rochester, Rochester, New York 14623 [R. C. R.]; University of Florida, College of Veterinary Medicine, Gainesville, Florida 32610 [L. E. F.]; Veterinary Cancer Referral Group, Los Angeles, California 90064 [M. R.]; University of Missouri, College of Veterinary Medicine, Columbia, Missouri 65211 [C. H.]; and Special Veterinary Services, Berkeley, California 94704 [J. F.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Spontaneous canine oral melanoma (COM) is a highly metastatic cancer, resistant to chemotherapy, and can serve as a model for cancer immunotherapy. Liposome-encapsulated muramyl tripeptide-phosphatidylethanolamine (L-MTP-PE) can activate the tumoricidal activity of the monocyte-macrophage system following i.v. injection. The objective of these studies was to evaluate the therapeutic effectiveness of L-MTP-PE administered alone and combined with recombinant canine granulocyte macrophage colony-stimulating factor (rcGM-CSF) in dogs undergoing surgery for oral melanoma.

Ninety-eight dogs with histologically confirmed, clinically staged, oral melanoma were entered into two randomized, double-blind, surgical adjuvant trials. In trial 1, 50 dogs were stratified based on clinical stage and randomized to once a week L-MTP-PE or lipid equivalent (control). When all of the clinical stages were combined, no difference in disease-free survival or in survival time (ST) were detected. However, within stage I, dogs receiving L-MTP-PE had a significant increase in ST compared with control, with 80% of the dogs treated with L-MTP-PE still alive at >2 years. Within each stage II and stage III, there was no difference detected between the treatment groups. In trial 2, 48 dogs were stratified on the basis of clinical stage and extent of surgery (simple resection or radical excision), treated with L-MTP-PE two times a week, and randomized to rcGM-CSF or saline (placebo) given s.c. daily for 9 weeks. Within each stage and when all of the stages were combined, 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 tumor location in the oral cavity, sex, type/extent of surgery, or age. In a subset of dogs tested, pulmonary alveolar macrophage cytotoxicity was enhanced with combined rcGM-CSF and L-MTP-PE but not in dogs treated with L-MTP-PE alone.

The present study indicates that after surgery, L-MTP-PE administered alone or combined with rcGM-CSF showed no significant antitumor activity in treating advanced stage COM. In early stage COM, L-MTP-PE was 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
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
There is ample evidence that the immune system can modulate the progression and metastasis of malignant melanoma. Several biological agents and approaches have demonstrated antitumor activity (1, 2, 3, 4, 5, 6, 7, 8, 9, 10) . The most widely used treatments include IFN-{alpha} (5 , 6) , IL-26 and adoptive immunotherapy (11) , and active immunization using tumor vaccines (12, 13, 14, 15) . Advances in melanoma treatment over the past decade have been directed at the role of adjuvant systemic therapy for patients with melanoma at high risk of recurrence or metastasis (1 , 9 , 10 , 16) .

Canine melanoma is a highly metastatic spontaneous tumor arising most commonly in the oral cavity (17, 18, 19) . COM has a biological behavior that is similar to oral melanoma in humans. Melanomas are the most common neoplasm seen in the oral cavity in dogs (20, 21, 22) . These tumors arise in many breeds but are more frequently identified in the dark pigmented breeds (e.g., Scottish terrier, Boston terrier, Airedale, and Cocker spaniel). COM occurs most often in dogs that are 8–12 years of age, roughly corresponding to the ages of highest prevalence in humans (40–60 years; Ref. 22 ). Metastasis is common and can be seen in regional lymph nodes, lung, liver, brain, and kidney. The treatment of choice is surgical excision, and, depending on clinical stage, the median ST is 8–10 months, with death due to 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 destroy a wide range of syngeneic, allogeneic, and xenogeneic tumors (25 , 26) . In rodents, dogs, and humans, a liposome-encapsulated lipophilic derivative of muramyl dipeptide, known as MTP-PE, when administered in vivo can activate monocytes and macrophages resulting in antitumor activity (27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38) . It has been shown that L-MTP-PE can result in the regression of established metastasis in the B16 murine melanoma model (39) . In humans with melanoma, two Phase II trials using L-MTP-PE have been reported (32 , 33) . In one study, 30 patients with measurable metastatic melanoma were treated, and, although no responses were detected, peripheral blood monocyte cytotoxicity was increased (32) . In the other study, 18 stage III melanoma patients with resectable tumors were treated with surgery and L-MTP-PE. After the administration of L-MTP-PE, enhanced monocyte cytotoxicity was detected in 6 of the 9 patients still 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) . Undoubtedly, 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 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) .

GM-CSF is a cytokine with a unique ability to stimulate differentiation of hematopoietic progenitor cells into dendritic cells, a potent antigen-presenting cell (47 , 48) . GM-CSF has received particular attention for enhancing the antitumor activity in transduced tumor cell vaccines (14 , 15 , 49) . In vitro, GM-CSF has been shown to slightly enhance cytotoxic activity of peripheral blood monocytes and lymphocytes and to markedly increase ADCC (50 , 51) and enhanced monocyte cytotoxicity against a melanoma cell line (52) . In vivo administration of GM-CSF in human cancer patients enhanced monocyte ADCC (53) and increased secretion of both TNF-{alpha} and IFN (54) . In another study in cancer patients, GM-CSF enhanced monocyte cytoxicity against colon HT29 tumor cells, although no antitumor responses were seen (55) .

This study was designed in a randomized manner to evaluate the efficacy of L-MTP-PE when used in a surgical adjuvant setting and administered alone or in combination with GM-CSF.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patient Population.
Ninety-eight dogs with previously untreated, histologically confirmed spontaneous oral melanoma, without radiographic evidence of distant metastasis, were studied. The pretreatment evaluation included a complete physical examination, three-dimensional measurements of the primary tumor, a complete blood count, serum chemistry profile, and urinalysis. For evaluation of metastatic disease, regional lymph nodes were evaluated with fine needle aspiration or biopsy and radiographs of the thorax. Only dogs without evidence of overt lung metastasis, in overall good health, and a tumor (including regional lymph nodes) amenable to complete surgical removal were eligible for entry into this study. Depending on the location of the primary tumor, radiographs of the mandible or maxilla were performed under general anesthesia to determine the extent 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 the primary tumor (stage I and II) and regional lymph nodes (stage III). For tumors involving the gingival region—especially if bone involvement was detected in the mandible or maxilla—a partial or complete mandibulectomy or partial maxillectomy was performed as described previously (56 , 57) . The decision to perform this procedure was at the discretion of the surgeon. All of the dogs entered were considered free of clinically detectable tumor after surgery. After surgery, all of the dogs were clinically staged, according to the WHO staging system, into stage I (tumors <2-cm diameter, node negative), stage II (2- to 5-cm diameter, node negative), or stage III (>5-cm diameter and/or node positive). This was a multicenter trial in which dogs from nine veterinary facilities were entered. One investigator (I. D. K.) performed all of the randomization. Drugs for each dog entered were prepared at the University of Wisconsin-Madison and sent by overnight express mail to the participating investigator. A schematic diagram of the protocols for the two trials is shown in Fig. 1Citation .



View larger version (15K):
[in this window]
[in a new window]
  		Fig. 1. Schematic diagram of the two oral melanoma clinical trials.

 
Trial 1.
After surgery and stratification based on clinical stage, dogs were treated in a double-blind fashion with L-MTP-PE or lipid equivalent (MLV) once a week for 8 weeks. The liposomes were given via a slow i.v. infusion over 5–8 min. The first dose of L-MTP-PE was 1 mg/m2; all of the subsequent doses were scaled according to the weight of the dog (1 mg/m2 for dogs <5 kg, 1.5 mg/m2 for dogs 5–10 kg, and 2 mg/m2 for dogs >10 kg). This dose of L-MTP-PE was similar to that used in previous studies (41, 42, 43) .

Trial 2.
After surgery, dogs were stratified by clinical stage. The extent of surgery was left to the discretion of the surgeon. To minimize potential bias related to the extent of surgery, dogs were further stratified on the basis of type/extent of surgery. Surgery was either radical excision (mandibulectomy or maxillectomy) versus simple excision (complete excision of tumor but not involving removal of bone). After stratification, the dogs were randomized to receive rcGM-CSF (initial dose 15 µg/kg) or saline, s.c. daily for 9 weeks. The frequency of administration of L-MTP-PE was increased from once a week (in trial 1) to two times a week using the same dosage as described for trial 1. L-MTP-PE was increased from once a week to two times a week because of the lack of therapeutic efficacy detected in stage II and stage III dogs in trial 1.

Liposome Preparation.
Lyophilized liposomes with or without MTP-PE (CGP 19835A lipid) were provided by Ciba-Geigy, Ltd. (Basel, Switzerland). Liposomes were prepared from a freeze-dried preparation by the addition of buffered saline, without calcium or magnesium, to vials containing dioleoyl-phosphatidylserine and 1-palmitoyl-2-oleoyl-phosphatidlycholine at a 3:7 molar ratio, with (L-MTP-PE) or without (MLV) MTP-PE. The ratio of active ingredient (MTP-PE) to phospholipid was 1:250. After 1 min, the vial contents were agitated on a vortex mixer or vigorously shaken by hand for 1 min, and then the contents were diluted with buffered saline to a concentration of 25 mg lipid/ml.

GM-CSF.
rcGM-CSF was kindly provided by Amgen, Inc. (Thousand Oaks, CA). Dogs were initially administered 15 µg/kg s.c. daily for 9 weeks, a dose that we found to enhance monocyte tumor cytostasis (58) and is well tolerated in normal experimental dogs. Dogs were started on rcGM-CSF or saline for 1 week prior to L-MTP-PE treatment and continued concurrently with L-MTP-PE for 8 weeks. The rationale to begin rcGM-CSF 1 week before L-MTP-PE was to allow for an increase in absolute monocyte count before the administration of L-MTP-PE. Because of toxicity in 10 dogs in the early phase of trial 2, the dose of rcGM-CSF was reduced to 5 µg/kg/day.

Follow-Up Evaluation.
In both trials, dogs were evaluated with routine physical examination and thoracic radiographs at 3-month intervals. Physical and historical abnormalities were investigated as clinically indicated at each recheck visit. Patient evaluations continued as long as necessary to determine DFS and overall ST for each dog. The DFS was defined as the time from surgery to evidence of clinical recurrence or metastasis. ST was defined as the time from surgery to death or euthanasia due to advanced disease. Euthanasia was performed when requested by the dog’s owner because the animal’s quality of life was considered severely limited due to advanced disease.

Cytotoxicity Assays.
A subset of dogs in trial 2 was tested to determine PAM cytotoxic activity after L-MTP-PE with or without rcGM-CSF. PAM cytotoxic activity was evaluated before rcGM-CSF or saline was administered, immediately prior to the first L-MTP-PE treatment and two days after the second L-MTP-PE treatment. Dogs were placed under general anesthesia and PAMs were collected via BAL, using a cold-sterilized fiberoptic bronchoscope and sterile saline according to the procedure described previously (59) . PAMs were isolated from lavage fluid using sterile, endotoxin-free density gradient separation (Ficoll-Hypaque, specific gravity 1.077, Sigma Chemical Company, St. Louis, MO). The coculture cytotoxicity assay used has been recently described in dogs (60) . Briefly, PAMs were added to wells of a 96-well microtiter plate and allowed to adhere for 90 min at 37°C in 5% CO2 humidified atmosphere. PAMs were cultured in RPMI 1640 (Life Technologies, Inc., Grand Island, NY) supplemented with 10% heat-inactivated FCS (Intergen, Purchase, NY), and 2 mM sodium pyruvate, 2 mML-glutamine, 10 mM HEPES buffer, 100 units/ml penicillin, and 100 µg/ml streptomycin (Sigma Chemical Company). After adherence, nonadherent PAMs were gently removed. To determine purity of the adherent cell population, cytospin preparations (Cytospin-2 Shandon Inc. Pittsburgh, PA) were made of the cells. The cytospin preparations were stained with Diff-Quick (American Scientific Products, McGaw Park, IL) and examined microscopically. After a 24-h incubation, 3H-labeled CML-6M-C2 canine melanoma cells (kindly provided by L. Wolfe, Auburn University, Auburn, AL) were added to each well in E:T ratios of 2.5:1, 5:1, and 10:1. Tumor cells were added to additional wells for the determination of spontaneous and maximal release of 3H. Cells were cocultured for 72 h, and then, the radioactivity (cpm) of 50 µl of supernatant from each well was determined. Percent cytotoxicity of CML-6M-C2 cells was calculated by the following formula:

where T = mean cpm of supernatant from wells containing tumor and effector cells, S (spontaneous release) = mean cpm of supernatant from wells containing tumor cells alone, and M (maximal release) = mean cpm of lysate from wells containing tumor cells alone.

Anesthesia for the BAL.
Dogs were premedicated with midazolam (0.2 mg/kg, maximum of 5 mg, s.c.) and butorphanol (0.2 mg/kg, maximum of 10 mg, s.c.) 30 min before anesthesia. Anesthesia was induced by injectable thiopentothal sodium (12–15 mg/kg) and maintained on isoflurane and 100% oxygen, using an endotracheal tube. A bronchoscope was passed through the larynx, and the BAL was performed as described previously (59) .

Statistical Analysis.
In designing trial 1 in dogs with oral melanoma, the expected median ST for surgery alone (all of the stages combined) is 8 months (18 , 23) ; we anticipated a 40% increase in the median survival in dogs treated with L-MTP-PE. With 25 dogs per arm, we should be able to detect that difference with an 80% power at the P = 0.05 level. For trial 2, we expected a median ST of 11 months for surgery and L-MTP-PE, and we anticipated a 40% increase in the median survival with the addition of rcGM-CSF. With 25 dogs per arm, we would be able to detect that difference with an 80% power at the P = 0.05 level.

DFS and overall ST were compared between the treatment groups of both trials. STs were also compared between dogs receiving L-MTP-PE once a week in trial 1 and dogs receiving L-MTP-PE two times a week in trial 2. Survival curves were generated by the Kaplan-Meier method and were compared using the Breslow and Mantel-Cox tests of significance between survival curves (61, 62, 63) . These statistical tests adjust for dogs still alive or lost to follow-up at the time of analysis. A P of less than 0.05 was considered significant.

Differences in percent cytoxicity were assessed using paired t tests. For results covering a range of E:T ratios, the highest P is reported.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Patient Characteristics.
Patient characteristics for all of the dogs entered into both trials are given in Table 1Citation .


View this table:
[in this window]
[in a new window]
  		Table 1 Patient characteristics

 
Treatment and Survival Data.
The L-MTP-PE treatments were well tolerated by the dogs. The only consistent side effect was an elevation in body temperature (1–2°C), lasting 1–4 h after the liposome treatment. The temperature elevation was more pronounced in the L-MTP-PE groups. Body temperature returned to normal within 6 h after injection. The fever response was most apparent on the first few treatments and then subsided on subsequent treatments. In trial 2, the initial dosage for rcGM-CSF was 15 µg/kg s.c. daily. However, 10 dogs experienced adverse side effects that included thrombocytopenia (n = 4), anterior uveitis (n = 2), lethargy and mild diarrhea (n = 2), gastritis (n = 1), and polydipsia/polyuria (n = 1). After documentation of these adverse side effects, all of the subsequent dogs were treated at a dose of 5 µg/kg. Table 2Citation summarizes the median DFS and ST for dogs according to clinical stage and treatment group for both trial 1 and 2.


View this table:
[in this window]
[in a new window]
  		Table 2 Analysis of DFS and overall STs

 
Trial 1.
Fifty dogs were entered into this clinical trial. Twenty-five were randomized to L-MTP-PE and 25 to MLV. When all of the stages were combined, there was no difference between dogs treated with L-MTP-PE versus placebo (Fig. 2)Citation . However, within stage I, dogs receiving L-MTP-PE had significantly prolonged ST (P < 0.05) compared with those receiving MLV (Fig. 3)Citation . In the L-MTP-PE group, 80% of the dogs were alive at 2 years compared with 25% in the MLV group. The statistical difference detected is based on 11 dogs treated with L-MTP-PE and 9 dogs treated with MLV (control). Increasing the patient population with stage I COM would increase the power of this observation. However, the 25% 2-year ST for the stage I dogs treated with MLV is similar to previous ST for dogs treated by surgery alone (23) .



View larger version (14K):
[in this window]
[in a new window]
  		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.

 


View larger version (13K):
[in this window]
[in a new window]
  		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 < 0.045. There was no significant difference between treatment groups with regard to DFS.

 
When treatment groups were combined, stage I (n = 20) had significantly prolonged (P < 0.022) DFS and ST compared with stage II (n = 19) and stage III (n = 11). The prognostic significance of the clinical staging system confirms similar findings in a previously published study (23) . No effect on DFS or ST was observed with regard to the location of the primary tumor, type of surgery, sex, or age (data not shown).

Trial 2.
Forty-eight dogs were entered into this clinical trial. All of the dogs received L-MTP-PE two time a week, and 24 were randomized to receive rcGM-CSF and 24 to receive saline. Within each stage and with all of the stages combined, there was no difference in DFS or ST when dogs receiving L-MTP-PE and rcGM-CSF were compared with dogs receiving L-MTP-PE and saline (Fig. 4)Citation . No effect on DFS or ST was observed with regard to the location of primary tumor, type of surgery, sex, or age (data not shown).



View larger version (13K):
[in this window]
[in a new window]
  		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.

 
Trials 1 and 2 Combined.
Comparison of dogs receiving L-MTP-PE once a week (trial 1) or two times a week (trial 2) showed no difference in DFS or ST with regard to frequency of L-MTP-PE (Fig. 5)Citation . Further analysis on type of surgery (simple versus radical), revealed no significant difference in DFS or ST (Table 2)Citation .



View larger version (16K):
[in this window]
[in a new window]
  		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.

 
Cytotoxicity Assays.
In 5 dogs, PAMs were collected by BAL and were assayed for cytotoxic activity against the canine melanoma cell line, CML-6M-C2, before and after rcGM-CSF (n = 3) or saline (n = 2) and L-MTP-PE. Mean (±SE) levels for percent cytotoxicity are shown in Fig. 6Citation . Cytotoxic activity was markedly increased (P = 0.053) in the rcGM-CSF group after receiving L-MTP-PE. At a 10:1 Et ratio for PAMs:tumor target, the pretreatment cytotoxicity against CML-6M-C2 was a mean of 13% and increased to 68% after rcGM-CSF and L-MTP-PE administration. In the group of dogs receiving L-MTP-PE plus saline, no increase in PAM cytotoxicity was detected (Fig. 6)Citation .



View larger version (12K):
[in this window]
[in a new window]
  		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 = 3) or saline (B, n = 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.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present surgical adjuvant therapy for stages I, II, and III (American Joint Committee on Cancer) melanoma is the use of high-dose IFN-{alpha} (10) . Before the approval of IFN-{alpha}, a large number of randomized trials evaluated the use of nonspecific bacteria immunostimulants in the surgical adjuvant setting. Bacille Calmette-Guerin, the bovine mycobacterium used as a tuberculosis vaccine, has been extensively tested (2 , 64 , 65) . A total of 13 published trials involving 1152 patients have been reported (10) . None of these randomized controlled studies has shown any significant impact on relapse-free or overall survival in the treated patients. A heat-killed preparation of Corynebacterium parvum (Propionobacterium acnes) has been shown to provide a slight survival advantage (3) ; however, other trials did not show any survival advantage (4 , 66 , 67) . One study using C. parvum showed a survival advantage in a surgical adjuvant trial in COM (23) . No randomized clinical trials have been conducted using adjuvant L-MTP-PE in human melanoma. The trials presented in this paper are the first randomized surgical adjuvant trials using L-MTP-PE in spontaneous malignant melanoma.

Patient characteristics for the population studied were similar to those reported elsewhere (17, 18, 19 , 23 , 68) . The results of these trials show that L-MTP-PE, when administered in a surgical adjuvant setting, has minimal antitumor activity, as evidenced in dogs with early stage (stage I) COM. Our study demonstrated a significant reduction in metastasis as evidenced by an increase in ST, with 80% having long-term survival (>2 years). However, this difference was seen only in stage I, and the number of dogs randomized within this stage was small (n = 20). Although only 9 dogs were treated with MLV (surgery alone), our previous studies have shown that stage I dogs with COM that are treated with surgery alone have a similar median ST (23) . In trial 2, all of the dogs were treated with L-MTP-PE, and the median ST for stage I dogs was 622 days, which suggests a greater therapeutic efficacy than other studies using surgery alone (18 , 23) .

An important change made between trial 1 and trial 2 was in increasing the administration of L-MTP-PE from once a week to two times a week. This change was made to determine whether two-times-a-week treatment would increase the therapeutic response in stage II and III dogs. Results from trial 2 demonstrated that increasing the treatment frequency to two times a week did not enhance the therapeutic activity of L-MTP-PE. In previous studies using rodent models, two-times-a-week treatments were selected because L-MTP-PE was shown to activate macrophage tumoricidal function for 48–72 h, and two-times-a-week treatment would maintain that activation (25 , 69) . In comparing the results of trials 1 and 2, no difference between once- or twice-weekly L-MTP-PE administration was seen in dogs with stage II or stage III disease.

Another interesting finding from this study was the demonstration that the extent of surgery (simple resection versus radical resection) had no significant influence on DFS and ST (Table 2)Citation . Other reported studies in COM using radical surgery alone (maxillectomy and mandibulectomy) report median STs of 4.5–10 months (56 , 57 , 70, 71, 72) . These STs compare favorably with the median STs reported in this study. This indicates that the major concern with COM is metastasis, and therapy must be directed toward metastasis control.

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. We have previously reported that PAMs, collected using BAL, can be activated in vitro using IFN-{gamma} (60 , 73) . We have found that the collection of PAM is a quick and convenient method to isolate large numbers of highly purified macrophages for immune testing. Although we did not show significant activation of tumor cell cytoxicity using rcGM-CSF alone, we did detect PAM cytotoxicity against the CML-6M-C2 (a cell line derived from a spontaneous canine melanoma metastasis; Ref. 74 ) after L-MTP-PE administration. We did not detect PAM cytoxicity in the 2 dogs tested after treatment with L-MTP-PE. In previous studies performed in our laboratory using clinically normal dogs, L-MTP-PE alone will increase PAM cytotoxicity (using an OSA tumor target) from 10% pretreatment to 35% posttreatment with L-MTP-PE (60) . Studies in dogs have shown that in vivo administration of L-MTP-PE will increase cytokine serum levels of TNF-{alpha} and IL-6, and blood monocyte cytotoxicity (34 , 35 , 37 , 38 , 46) . In human patients, L-MTP-PE has been shown to activate blood monocytes and increase serum TNF-{alpha}, IL-6, neopterin, and IL-1{beta}. However, no correlation has been found between these cytokines and clinical response to L-MTP-PE (27 , 31, 32, 33 , 41 , 75) .

Cytokines such as IFN, IL-2, IL-4, and GM-CSF can up-regulate antigen expression in melanoma cells, enhance helper T-cell function, and augment antigen presentation. GM-CSF can activate neutrophils, eosinophils, and macrophages to lyse tumor cells directly or to mediate ADCC (51) . It can also increase MHC expression of tumor antigen to immune effector cells inasmuch as GM-CSF is important in the maturation of bone marrow-derived dendritic cells (48 , 76) . Our rationale for combining systemic GM-CSF with L-MTP-PE was 3-fold: (a) to increase the monocyte/macrophage population; (b) to enhance monocyte/macrophage tumoricidal activity; and (c) to increase antigen presentation by dendritic cells, the most potent antigen-presenting cells in the immune system. However, it has also been reported that GM-CSF may activate suppressor mechanisms to regulate the antitumor response negatively (77 , 78) or enhance the tumorigenicity of some tumor cells (79 , 80) . Our study did not demonstrate any therapeutic advantage of rcGM-CSF over L-MTP-PE alone. However, the total number of dogs entered (n = 48) into trial 2 may have been too small to detect a difference between each treatment group.

In experimental dogs, doses of 50 µg/kg/day have been studied for hematological activity, and the most common side effect reported is thrombocytopenia due to activation of the monocyte-macrophage system resulting in a decrease in platelet survival (81 , 82) . In this study, we selected a dose of 5 µg/kg (150 µg/m2 s.c. daily) because of toxicity detected at 15 µg/kg (450 µg/m2). In human clinical trials, the doses tested for GM-CSF (when used systemically to activate monocytes or to enhance ADCC) have ranged from 15 to 500 µg/m2/day (48 , 54 , 55 , 83) with 150–250 µg/m2 being the most effective dose.

In summary, L-MTP-PE administered alone or combined with rcGM-CSF has minimal antitumor activity when administered in a surgical adjuvant setting. However, there is suggestive evidence that L-MTP-PE results in the prolongation of survival in dogs with early (stage I) malignant COM. Furthermore, this study helps to strengthen the rationale for the use of the dog as a model for human spontaneous malignant melanoma.


   ACKNOWLEDGMENTS
 
We thank W. C. Kisseberth, C. A. London, L. Kravis, R. Chun, D. Thamm, E. Hershey, G. S. Hogge, and S. C. Helfand for participation in patient management.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by a grant from the Morris Animal Foundation, Englewood, CO, and Ciba-Geigy Ltd., Basel, Switzerland. Back

2 To whom requests for reprints should be addressed, at Department of Medical Sciences, University of Wisconsin, 2015 Linden Drive West, Madison, WI 53706. Back

3 Present address: University of Texas, M. D. Anderson Cancer Center, 1515 Holcombe Boulevard., Houston, TX 77054. Back

4 Present address: Southwest Surgery and Oncology, 17672 Cowan Avenue, Irvine, CA 92614. Back

5 Present address: University of Zurich, Winterthurerstrasse 260, CH-8057 Zurich, Switzerland. Back

6 The abbreviations used are: IL, interleukin; DFS, disease-free survival; ST, survival time; PAM, pulmonary alveolar macrophage; MTP-PE, muramyl tripeptide-phosphatidylethanolamine; L-MTP-PE, liposome-encapsulated MTP-PE; MLV, multilamellar vesicle; OSA, osteosarcoma; GM-CSF, granulocyte macrophage colony-stimulating factor; rcGM-CSF, recombinant canine GM-CSF; ADCC, antibody-dependent cellular cytotoxicity; TNF, tumor necrosis factor; COM, canine oral melanoma; BAL, bronchoalveolar lavage. Back

Received 3/ 4/99; revised 8/30/99; accepted 9/21/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Johnson T. M., Yahanda A. M., Chang A. E., Fader D. J., Sondak V. K. Advances in melanoma therapy. J. Am. Acad. Dermatol., 38: 731-741, 1998.[Medline]
  2. Veronesi U., Adamus J., Aubert C., Bajetta E., Beretta G., Bonadonna G., Bufalino R., Cascinelli N., Cocconi G., Durand J., De Marsillac J., Ikonopisov R. L., Kiss B., Lejeune F., MacKie R., Madej G., Mulder H., Mechl Z., Milton G. W., Morabito A., Peter H., Priario J., Paul E., Rumke P., Sertoli R., Tomin R. A. A randomized trial of adjuvant chemotherapy and immunotherapy in cutaneous melanoma. N. Engl. J. Med., 307: 913-916, 1982.[Abstract]
  3. Lipton A., Harvey H. A., Lawrence B., Gottlieb R., Kukrika M., Dixon R., Graham W., Miller S., Heckard R., Schelzel D., White D. S. Corynebacterium parvum versus BCG adjuvant immunotherapy in human malignant melanoma. Cancer (Phila.), 51: 57-60, 1983.[Medline]
  4. Lipton A., Harvey H. A., Balch C. M., Antle C. E., Heckard R., Bartolucci A. A. Corynebacterium parvum versus Bacille Calmette-Guerin adjuvant immunotherapy of stage II malignant melanoma. J. Clin. Oncol., 9: 1151-1156, 1991.[Abstract]
  5. Creagan E. T., Dalton R. J., Ahmann D. L., Jung S. H., Morton R. F., Langdon R. M., Jr., Kugler J., Rodrigue L. J. Randomized, surgical adjuvant clinical trial of recombinant interferon {alpha}-2a in selected patients with malignant melanoma. J. Clin. Oncol., 13: 2776-2783, 1995.[Abstract]
  6. Kirkwood J. M., Strawderman M. H., Ernstoff M. S., Smith T. J., Bordon E. C., Blum R. H. Interferon {alpha}-2b adjuvant therapy of high-risk resected cutaneous melanoma: the Eastern Cooperative Oncology Group trial EST 1684. J. Clin. Oncol., 14: 7-17, 1996.[Abstract]
  7. Chang A. E., Sondak V. K., Bishop D. K., Nickoloff B. J., Mulligan R. C., Mule J. J. Adoptive immunotherapy of cancer with activated lymph node cells primed in vivo with autologous tumor cells transduced with the GM-CSF gene. Hum. Gene Ther., 7: 773-792, 1996.[Medline]
  8. Rosenberg S. A. Development of cancer immunotherapies based on identification of the genes encoding cancer regression antigens. J. Natl. Cancer Inst., 88: 446-453, 1996.
  9. Ollila D. W., Kelley M. C., Gammon G., Morton D. L. Overview of melanoma vaccines: active specific immunotherapy for melanoma patients. Semin. Surg. Oncol., 14: 328-336, 1998.[Medline]
  10. Agarwala S. S., Kirkwood J. M. Adjuvant therapy of melanoma. Semin. Surg. Oncol., 14: 302-310, 1998.[Medline]
  11. Rosenberg S. A., Yannelli J. R., Yang J. C., Topalian S. L., Schwartzentruber D. J., Weber J. S., Parkinson D. R., Seipp C. A., Einhorn J. H., White D. E. Treatment of patients with metastatic melanoma with autologous tumor-infiltrating lymphocytes and interleukin 2. J. Natl. Cancer Inst., 86: 1159-1166, 1994.[Abstract/Free Full Text]
  12. Livingston P. O., Wong G. Y., Adluri S., Tao Y., Padavan M., Parente R., Hanlon C., Calves M. J., Helling F., Ritter G. Improved survival in stage III melanoma patients with GM2 antibodies: a randomized trial of adjuvant vaccination with GM2 ganglioside. J. Clin. Oncol., 12: 1036-1044, 1994.[Abstract/Free Full Text]
  13. van der Bruggen P., Bastin J., Gajewski T., Coulie P. G., Boel P., De Smet C., Traversari C., Townsend A., Boon T. A peptide encoded by human gene MAGE-3 and presented by HLA-A2 induces cytolytic T lymphocytes that recognize tumor cells expressing MAGE-3. Eur. J. Immunol., 24: 3038-3043, 1994.[Medline]
  14. Hogge G. S., Burkholder J. K., Culp J., Albertini M. R., Dubielzig R. R., Keller E. T., Yang N.-S., MacEwen E. G. Development of human granulocyte-macrophage-colony stimulating factor-transfected tumor cell vaccines for the treatment of spontaneous canine cancer. Hum. Gene Ther., 9: 1851-1861, 1998.[Medline]
  15. Soiffer R., Lynch T., Mihm M., Jung K., Rhuda C., Schmollinger J. C., Hodi F. S., Liebster L., Lam P., Mentzer S., Singer S., Tanabe K. K., Cosimii A. B., Duda R., Sober A., Bhan A., Daley J., Neuberg D., Parry G., Rokovich J., Richards L., Drayer J., Berns A., Clift S., Dranoff G. Vaccination with irradiated autologous melanoma cells engineered to secrete human granulocyte-macrophage colony stimulating factor generates potent antitumor immunity in patients with metastatic melanoma. Proc. Natl. Acad. Sci. USA, 95: 13141-13146, 1998.[Abstract/Free Full Text]
  16. Grob J. J., Dreno B., Salmoniere P., Delaunay M., Cupissol D., Guillot B., Souteyrand P., Sassolas B., Cesarini J.-P., Lionnet S., Chastang C., Bonerandi J. J. Randomized trial of interferon {alpha}-2a as adjuvant therapy in resected primary melanoma thicker than 1.5 mm without clinically detectable node metastasis. Lancet, 351: 1905-1910, 1998.[Medline]
  17. Bostock D. E. Prognosis after surgical excision of canine melanomas. Vet. Pathol., 16: 32-40, 1979.[Abstract]
  18. Harvey H. J., MacEwen E. G., Braun D., Patnaik A. K., Withrow S. J., Jongeward S. Prognostic criteria for dogs with oral melanoma. J. Am. Vet. Med. Assoc., 178: 580-582, 1981.[Medline]
  19. Bateman K. E., Catton P. A., Pennock P. W., Kruth S. A. 0–7-21 radiation therapy for the treatment of canine oral melanoma. J. Vet. Internal Med., 8: 267-272, 1994.[Medline]
  20. Dorn C. R., Priester W. A. Epidemiologic analysis of oral and pharyngeal cancer in dogs, cats, horses, and cattle. J. Am. Vet. Med. Assoc., 169: 1202-1205, 1976.[Medline]
  21. Todoroff R. J., Brodey R. B. Oral and pharyngeal neoplasia in the dog: a retrospective survey of 361 cases. J. Am. Vet. Med. Assoc., 175: 567-571, 1979.[Medline]
  22. Patronek G. J., Waters D. J., Glickman L. T. Comparative longevity of pet dogs and humans: implications for gerontology research. J. Gerontol. A Biol. Sci. Med. Sci., 52: B171-B178, 1997.[Abstract]
  23. MacEwen E. G., Patnaik A. K., Harvey H. J., Hayes A. A., Matus R. Canine oral melanoma: comparison of surgery versus surgery plus Corynebacterium parvum. Cancer Invest., 4: 397-402, 1986.[Medline]
  24. Salisbury S. K., Lantz G. C. Long-term results of partial mandibulectomy for treatment of oral melanoma in 30 dogs. J. Am. Anim. Hosp. Assoc., 24: 285-294, 1988.
  25. Fidler I. J. Therapy of cancer metastasis by systemic activation of macrophages. Adv. Pharmacol., 30: 271-326, 1994.
  26. Fidler I. J. Recognition and destruction of target cells by tumoricidal macrophages. Isr. J. Med. Sci., 14: 177-191, 1978.[Medline]
  27. Kleinerman E. S., Erickson K. L., Schroit A. J., Fogler W. E., Fidler I. J. Activation of tumoricidal properties in human blood monocytes by liposomes containing lipophilic muramyl tripeptide. Cancer Res., 43: 2010-2014, 1983.[Abstract/Free Full Text]
  28. Philips N. C., Tsao M. Inhibition of experimental liver tumor growth in mice by liposomes containing a lipophilic muramyl dipeptide. Cancer Res., 49: 936-940, 1984.[Abstract/Free Full Text]
  29. Talmadge J. E., Lenz B. F., Klabansky R., Simon R., Riggs C., Guo S., Oldham R. K. Therapy of autochthonous skin cancers in mice with intravenously injected liposomes containing muramyl tripeptide. Cancer Res., 46: 1160-1163, 1986.[Abstract/Free Full Text]
  30. Saiki I., Milas L., Hunter N., Fidler I. J. Treatment of experimental lung metastasis with local thoracic irradiation followed by systemic activation with liposomes containing muramyl tripeptide. Cancer Res., 46: 4966-4970, 1986.[Abstract/Free Full Text]
  31. Kleinerman E. S., Murray J. L., Synder J. S., Cunningham J. E., Fidler I. J. Activation of tumoricidal properties in monocytes from cancer patients following intravenous administration of liposomes containing tripeptide phosphatidylethanolamine. Cancer Res., 49: 4665-4770, 1989.[Abstract/Free Full Text]
  32. Liebes L., Walsh C. M., Chachaua A., Oratz R., Richards D., Hochster H., Peace D., Marino D., Alba S., Sher D. L., Blum R. H., Vilcek J. Modulation of monocyte functions by muramyl tripeptide phosphatidylethanolamine in a Phase II study in patients with metastatic melanoma. J. Natl. Cancer Inst., 84: 694-699, 1992.[Abstract/Free Full Text]
  33. Fujimaki W., Itoh K., An T., Gano J. B., Ross M. I., Mansfield P. F., Balch C. M., Augustus L. B., Karkevitch D., Johnston D., Fidler I. J., Kleinerman E. S. Cytokine production and immune cell activation in melanoma patients treated with liposomal muramyl tripeptide (CGP 19835A Lipid). Cancer Biother., 8: 307-318, 1993.[Medline]
  34. Shi F., MacEwen E. G., Kurzman I. D. In vitro and in vivo effect of doxorubicin combined with liposome-encapsulated muramyl tripeptide on canine monocyte activation. Cancer Res., 53: 3986-3991, 1993.[Abstract/Free Full Text]
  35. Shi F., Kurzman I. D., MacEwen E. G. In vitro and in vivo production of interleukin-6 induced by muramyl peptides and lipopolysaccharide in normal dogs. Cancer Biother., 10: 317-325, 1995.[Medline]
  36. Kleinerman E. S., Jia S. F., Griffin J., Seibel N. L., Benjamin R., Jaffe N. Phase II study of liposomal muramyl tripeptide in osteosarcoma: the cytokine cascade and monocyte activation following administration. J. Clin. Oncol., 10: 1310-1316, 1992.[Abstract/Free Full Text]
  37. Kurzman I. D., Shi F., MacEwen E. G. In vitro and in vivo canine mononuclear cell production of tumor necrosis factor induced by muramyl peptides and lipopolysaccharide. Vet. Immunol. Immunopathol., 38: 45-56, 1993.[Medline]
  38. Smith B. W., Kurzman I. D., Schultz K. T., Czuprynski C. J., MacEwen E. G. Muramyl peptides augment the in vitro and in vivo cytostatic activity of canine plastic-adherent mononuclear cells against canine osteosarcoma cells. Cancer Biother., 8: 137-144, 1993.[Medline]
  39. Fidler I. J. Optimization and limitations of systemic treatment of murine melanoma metastasis with liposomes containing muramyl tripeptide phosphatidylethanolamine. Immunol. Immunother., 21: 169-173, 1986.
  40. Gianan M., Kleinerman E. S. Liposomal muramyl tripeptide (CGP 19835A lipid) therapy for resectable melanoma in patients who were at high risk for relapse: an update. Cancer Biother. Radiopharm., 13: 363-368, 1998.[Medline]
  41. Kleinerman E. S., Raymond A. K., Bucana C. D., Jaffe N., Harris M. B., Krakoff I. H., Benjamin R., Fidler I. J. Unique histological changes in lung metastases of osteosarcoma patients following therapy with liposomal muramyl tripeptide (CGP 19835A lipid). Cancer Immunol. Immunother., 34: 211-220, 1992.[Medline]
  42. Kleinerman E. S., Gano J. B., Johnston D. A., Benjamin R. S., Jaffe N. Efficacy of liposomal muramyl tripeptide (CGP 19835A) in the treatment of relapsed osteosarcoma. Am. J. Clin. Oncol., 18: 93-99, 1995.[Medline]
  43. MacEwen E. G. Spontaneous tumors in dogs and cats: models for the study of cancer biology and treatment. Cancer Metastasis Rev., 9: 125-136, 1990.[Medline]
  44. MacEwen E. G., Kurzman I. D., Rosenthal R. C., Smith B. W., Manley P. A., Roush J. K., Howard P. E. Therapy for osteosarcoma in dogs with intravenous injection of liposome-encapsulated muramyl tripeptide. J. Natl. Cancer Inst., 81: 935-938, 1989.[Abstract/Free Full Text]
  45. Kurzman I. D., MacEwen E. G., Rosenthal R. C., Fox L. E., Keller E. T., Helfand S. C., Vail D. M., Dubielzig R. R., Madewell B. R., Rodriguez C. O., Jr., Obradovich J., Fidel J., Rosenberg M. Adjuvant therapy for osteosarcoma in dogs: results of randomized clinical trials using combined liposome-encapsulated muramyl tripeptide and cisplatin. Clin. Cancer Res., 1: 1595-1601, 1995.[Abstract]
  46. Vail D. M., MacEwen E. G., Kurzman I. D., Dubielzig R. R., Helfand S. C., Kisseberth W. C., London C. A., Obradovich J. E., Madewell B. R., Rodriguez C. O., Jr., Fidel J., Susaneck S., Rosenberg M. Liposome-encapsulated muramyl tripeptide phosphatidylethanolamine adjuvant immunotherapy for splenic hemangiosarcoma in the dog: a randomized multi-institutional clinical trial. Clin. Cancer Res., 1: 1165-1170, 1995.[Abstract]
  47. Herbst B., Kohler G., Mackensen A., Veelken H., Lindemann A. GM-CSF promotes differentiation of a precursor cell of monocytes and Langerhans-type dendritic cells from CD34+ haematopoietic progenitor cells. Br. J. Haematol., 101: 231-241, 1998.[Medline]
  48. Armitage J. Emerging applications of recombinant human granulocyte-macrophage colony-stimulating factor. Blood, 92: 4491-4508, 1998.[Free Full Text]
  49. Dranoff G., Jaffe E., Lazenby A., Golumbek P., Levitsky H., Brose K., Jackson V., Hamada H., Pardoll D., Mulligan R. C. Vaccination with irradiated tumor cells engineered to secrete murine granulocyte-macrophage colony stimulating factor stimulates potent, specific, and long-lasting anti-tumor immunity. Proc. Natl. Acad. Sci. USA, 90: 3539-3543, 1993.[Abstract/Free Full Text]
  50. Masucci G., Wersall P., Ragnhammar P., Mellstedt H. Granulocyte-monocyte-colony-stimulating factor augments the cytotoxic capacity of lymphocytes and monocytes in antibody dependent cellular cytotoxicity. Cancer Immunol. Immunother., 29: 288-292, 1989.[Medline]
  51. Ragnhammar P., Frodin J-E., Trotta P. P., Mellstedt H. Cytotoxicity of white blood cells activated by granulocyte-macrophage-colony-stimulating factor against tumor cells in the presence of various antibodies. Cancer Immunol. Immunother., 39: 254-262, 1994.[Medline]
  52. Grabstein K. H., Urdal D. L., Tushinski R. J., Mochizuki D. Y., Price V. L., Cantrell M. A., Gillis S., Conlon P. J. Induction of macrophage tumoricidal activity by granulocyte-macrophage colony-stimulating factor. Science (Washington DC), 232: 506-508, 1986.[Abstract/Free Full Text]
  53. Chachoua A., Oratz R., Liebes L., Alter R., Felice A., Pease D., Vilcek J., Blum R. Phase Ib trial for granulocyte-macrophage colony-stimulating factor combined with murine monoclonal antibody R24 in patients with malignant melanoma. J. Immunother. Emphasis Tumor Biol., 16: 132-141, 1994.
  54. Wing E., Magee D., Whiteside T., Kaplan S., Shadduck R. Recombinant human granulocyte-macrophage colony-stimulating factor enhances monocyte cytotoxicity and secretion of tumor necrosis factor {alpha} and interferon in cancer patients. Blood, 73: 643-646, 1989.[Abstract/Free Full Text]
  55. Chachoua A., Oratz R., Hoogmoed R., Caron D., Pease D., Liebes L., Blum R., Vilcek J. Monocyte activation following systemic administration of granulocyte-macrophage colony-stimulating factor. J. Immunother., 15: 217-224, 1994.[Medline]
  56. White R. Mandibulectomy and maxillectomy in the dog: long-term survival in 100 cases. J. Small Anim. Pract., 32: 69-74, 1991.
  57. Kosovsky J., Matthiesen D. T., Marretta S. M., Patnaik A. K. Results of partial mandibulectomy for the treatment of oral tumors in 142 dogs. Vet. Surg., 20: 397-401, 1991.[Medline]
  58. Kurzman I. D., MacEwen E. G., Broderick C., Witte P. Effect of colony-stimulating factors on number and function of circulating monocytes in normal dogs. Mol. Biother., 4: 29-33, 1992.[Medline]
  59. Vail D. M., Mahler P., Soergel S. Differential cell analysis including phenotypic subtype of lymphocytes in bronchoalveolar lavage fluid from clinically normal dogs. Am. J. Vet. Res., 56: 282-285, 1995.[Medline]
  60. Kurzman I. D., Shi F., Vail D. M., MacEwen E. G. In vitro and in vivo enhancement of canine pulmonary alveolar macrophage cytotoxic activity against canine osteosarcoma cells. Cancer Biother. Radiopharm., 14: 121-128, 1999.[Medline]
  61. Kaplan E. L., Meier P. Nonparametric estimation from incomplete observations. J. Am. Stat. Assoc., 53: 457-481, 1958.
  62. Breslow N. A. A generalized Kruskal-Wallis test for comparing samples subject to unequal patterns of censorship. Biometrika, 57: 579-594, 1970.[Abstract/Free Full Text]
  63. Cox D. R. Regression models and life tables. J. R. Stat. Soc. (Series B), 34: 187-220, 1972.
  64. Gutterman J. U., Mavligit G., McBride C., Frei E., III, Freireich E. J., Hersh E. M. Active immunotherapy with B. C. G. for recurrent malignant melanoma. Lancet, 1: 1208-1212, 1973.[Medline]
  65. Czarnetzki B. M., Macher E., Suciu S., Thomas D., Steerenberg P. A., Rumke P. Long-term adjuvant immunotherapy in stage I high risk malignant melanoma, comparing two BCG preparations versus non-treatment in a randomised multicentre study (EORTC Protocol 18781). Eur. J. Cancer, 29A: 1237-1242, 1993.
  66. Balch C. M., Murray D. R., Presant C., Sugarbaker E. V., Bartolucci A. A. A randomized prospective comparison of BCG versus C. parvum adjuvant immunotherapy in human patients with resected metastatic lymph nodes. Proc. Am. Soc. Clin. Oncol., 3: 263 1984.
  67. Balch C. M., Smalley R. V., Bartolucci A. A., Burns D., Presant C. A., Durant J. R. A randomized prospective clinical trial of adjuvant C. parvum immunotherapy in 260 patients with clinically localized melanoma (stage I): prognostic factors analysis and preliminary results of immunotherapy. Cancer (Phila.), 49: 1079-1084, 1982.[Medline]
  68. Dow S. W., Elmslie R. E., Willson A. P., Roche L., Gorman C., Potter T. A. In vivo tumor transfection with superantigen plus cytokine genes induces tumor regression and prolongs survival in dogs with malignant melanoma. J. Clin. Invest., 101: 2406-2414, 1998.[Medline]
  69. Fidler I. J., Sone S., Fogler W. E., Smith D., Braun D. G., Tarcsay L., Gisler R. J., Schroit A. J. Efficacy of liposomes containing a lipophilic muramyl dipeptide for activating the tumoricidal properties of alveolar macrophages in vivo. J. Biol. Response Modif., 1: 43-55, 1982.
  70. Wallace J., Matthiesen D. T., Patnaik A. K. Hemimaxillectomy for the treatment of oral tumors in 69 dogs. Vet. Surg., 21: 337-341, 1992.[Medline]
  71. Schwarz P., Withrow S. J., Curtis C. R., Powers B. E., Straw R. C. Mandibular resection as a treatment for oral cancer in 81 dogs. J. Am. Anim. Hosp. Assoc., 27: 601-610, 1991.
  72. Schwarz P., Withrow S. J., Curtis C. R., Powers B. E., Straw R. C. Partial maxillary resection as a treatment for oral cancer in 61 dogs. J. Am. Anim. Hosp. Assoc., 27: 617-624, 1991.
  73. Soergel, S., MacEwen, E. G., Vail, D. M., Potter, D., Sondel, P., and Helfand, S. C. The immunotherapeutic potential of activated canine alveolar macrophages and antitumor monoclonal antibodies in metastatic canine melanoma. J. Immunother., in press, 1999.
  74. Wolfe L., Oliver J., Smith B., Wolfe L. G., Oliver J. L., Smith B. B., Tovio-Kinnucan M. A., Powers R. D., Brawner W. R., Henderson R. A., Hankes G. H. Biologic characterization of canine melanoma lines. Am. J. Vet. Res., 48: 1642-1648, 1987.[Medline]
  75. Asano T., McIntyre B. W., Bednarczyk J. L., Wygant J. N., Kleinerman E. S. Liposomal muramyl tripeptide upregulates adhesion molecules on the surface of human monocytes. Oncol. Res., 7: 253-257, 1995.[Medline]
  76. Cella M., Engering A., Pinet V., Peters J., Lanzavecchia A. Inflammatory stimuli induce accumulation of MHC class II complexes on dendritic cells. Nature (Lond.), 388: 782-787, 1997.[Medline]
  77. Pak A. S., Ip G., Wright M. A., Young R. I. Treating tumor bearing mice with low-dose {gamma} interferon plus tumor necrosis factor-{alpha} to diminish immune suppressive granulocyte-macrophage progenitor cells increases responsiveness to interleukin-2 immunotherapy. J. Immunol., 153: 885-890, 1995.
  78. Young R. I., Wright M. A., Lozano Y., Matthews J. P., Benefield J., Prechel M. M. Mechanisms of immune suppression in patients with head and neck cancer: influence on the immune infiltrate of the cancer. Int. J. Cancer, 67: 333-338, 1996.[Medline]
  79. Tsuruta N., Yatsunami J., Takayama K., Nakanishi Y., Ichinose Y., Hara N. Granulocyte-macrophage colony stimulating factor stimulates tumor invasiveness in squamous cell lung carcinoma. Cancer (Phila.), 82: 2173-2183, 1998.[Medline]
  80. Wang J., Yang W. K., Yang Y., Wei S-J., Yang D-M., Whang-Peng J., Chen Y-M., Ting K-L., Ting C-C. Paradoxical effect of GM-CSF gene transfer on the tumorigenicity and immunogenicity of murine tumors. Int. J. Cancer, 75: 459-466, 1998.[Medline]
  81. Nash R. A., Schuening F., Applelbaum F., Hammond W. P., Boone T., Morris C. F., Slichter S. J., Storb R. Molecular cloning and in vivo evaluation of canine granulocyte-macrophage colony-stimulating factor. Blood, 78: 930-937, 1991.[Abstract/Free Full Text]
  82. Nash R. A., Burstein S. A., Storb R., Yang W., Abrams K., Appelbaum F. R., Boone T., Deeg H. J., Durack L. D., Schuening F. G., McDonough S., Moore P., Nelp W. B., Slichter S. Thrombocytopenia in dogs induced by granulocyte-macrophage colony-stimulating factor: increased destruction of circulating platelets. Blood, 86: 1765-1775, 1995.[Abstract/Free Full Text]
  83. Kleinerman E. S., Knowles R., Lachman L., Gutterman J. Effect of recombinant granulocyte/macrophage colony-stimulating factor on human monocyte activity in vitro and following intravenous administration. Cancer Res., 48: 2604-2609, 1988.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Leukoc. Biol.Home page
R. S. Kornbluth and G. W. Stone
Immunostimulatory combinations: designing the next generation of vaccine adjuvants
J. Leukoc. Biol., November 1, 2006; 80(5): 1084 - 1102.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
P. J. Bergman, J. McKnight, A. Novosad, S. Charney, J. Farrelly, D. Craft, M. Wulderk, Y. Jeffers, M. Sadelain, A. E. Hohenhaus, et al.
Long-Term Survival of Dogs with Advanced Malignant Melanoma after DNA Vaccination with Xenogeneic Human Tyrosinase: A Phase I Trial
Clin. Cancer Res., April 1, 2003; 9(4): 1284 - 1290.
[Abstract] [Full Text] [PDF]

Home page
 Vet PatholHome page
J. A. Ramos-Vara, M. E. Beissenherz, M. A. Miller, G. C. Johnson, L. W. Pace, A. Fard, and S. J. Kottler
Retrospective Study of 338 Canine Oral Melanomas with Clinical, Histologic, and Immunohistochemical Review of 129 Cases
Vet. Pathol., November 1, 2000; 37(6): 597 - 608.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by MacEwen, E. G.
Right arrow Articles by Fidel, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by MacEwen, E. G.