Expression of leptin and iNOS in oral melanomas in dogs.

BACKGROUND: Oral melanoma (OM) in dogs is an aggressive malignancy, with clinical behavior resembling cutaneous melanomas in humans. Melanoma in humans is promoted by an inflammatory environment that is contributed to by leptin and inducible nitric oxide synthase (iNOS). OBJECTIVE: To determine if the patterns of leptin and iNOS expression are similar in OM in dogs and cutaneous melanomas in humans. ANIMALS: Twenty client-owned dogs. METHODS: Retrospective case study. Immunostaining of the OM tumors from each dog was scored for percentage and intensity of leptin and iNOS expression. Mitotic index was used as an indicator of tumor aggression. RESULTS: Leptin was detected in ≥75% of the tumor cells in specimens from 11 dogs. One tumor expressed leptin in ≤25% of the cells. The intensity of leptin expression was variable with 6, 9, and 5 cases exhibiting low-, moderate-, and high-intensity staining, respectively. OM with the lowest percentage of iNOS positive cells displayed the highest mitotic indices (P = .006, ANOVA). CONCLUSIONS AND CLINICAL IMPORTANCE: The expression of leptin is a common finding in melanomas in dogs. These data suggest that the possibility of future clinical applications, such as measuring the concentrations of plasma leptin as a screening tool or leptin as a target for therapy. The relevance of iNOS is not as clear in dogs with OM, for which other directed therapeutics might be more appropriate.

Regulation of iNOS by the p44/42 mitogen-activated protein kinase pathway in human melanoma.

Activating mutations of the genes for NRAS and BRAF, components of the p44/42 mitogen-activated protein kinase (MAPK) pathway, are common findings in melanoma. Recent evidence in several nonmelanoma cell systems supports the regulation of the inducible nitric oxide synthase (iNOS) gene by this pathway. On the basis of our data showing that melanoma iNOS expression predicts shortened patient survival, we formulated the hypothesis that activating mutations of NRAS or BRAF, which lead to constitutive activation of the p44/42 MAPK pathway, drive iNOS expression in human melanoma. In the present study, we have shown that inhibition of melanoma iNOS activity by S-methylisothiourea leads to decreased cell proliferation, confirming the importance of iNOS activity for melanoma cell growth. Regulation of melanoma iNOS expression by the p44/42 MAPK pathway was demonstrated by inhibition of the pathway by U0126, and by BRAF RNA interference. To explore this regulatory pathway in human tissue, 20 melanoma tumors were examined for NRAS and BRAF mutations, immunohistochemical evidence of ERK phosphorylation, and iNOS expression. A significant association was found among these three features. We conclude that in human melanoma, activating mutations of NRAS and BRAF drive constitutive iNOS expression and, implicitly, nitric oxide production, contributing to the poor survival of these patients.

Autoimmunity and hypothyroidism in patients with uveal melanoma.

Among patients with advanced melanoma, the development of autoimmune phenomena or of hypothyroidism during therapy has been associated with a favourable outcome. The objective of this study was to determine the prevalence of autoimmunity and of hypothyroidism in the melanoma population as a whole and to determine if these disease states confer a survival advantage for patients with metastatic disease. We report our findings in the uveal melanoma population. The study population (n = 91) consisted of all patients registered at this institution with the diagnosis of uveal melanoma during a 2 year study period. Eight (8.8%) had a systemic autoimmune disease; 12 (13.2%) were hypothyroid, including 9/46 (19.6%) females. Survival of the stage 4 patients was determined from diagnosis of the primary tumour (SvDx) and from diagnosis of metastatic disease (SvMt), and was compared to that of age/sex matched stage 4 controls. For autoimmune patients versus controls, the median SvDx was 111 months vs 37 months (P = 0.2734) and the median SvMt was 17 months vs 4 months (P = 0.0887). For the hypothyroid patients versus controls, the median SvDx was 58 months vs 49 months (P = 0.5348) and the median SvMt was 4 months vs 8 months (P = 0.2437). We conclude that there is a trend toward longer survival from the date of metastasis in uveal melanoma patients with a systemic autoimmune disorder, suggesting that systemic autoimmunity may play a role in modifying the activity of established metastases. This trend is not seen among the uveal melanoma patients with hypothyroidism. The high prevalence of hypothyroidism suggests a possible molecular interaction between the two disease processes.

A phase II study of "decrescendo" interleukin-2 plus interferon-alpha-2a in patients with progressive metastatic melanoma after chemotherapy.

BACKGROUND: The authors tested a biotherapy regimen involving recombinant interferon-alpha-2a (rIFN-alpha-2a) and recombinant human interleukin-2 (rhIL-2), given in a "decrescendo" schedule over 5 days, for its activity and toxicity in 21 patients who previously had received chemotherapy for advanced melanoma. METHODS: Patients (15 men and 6 women) were given intravenous rhIL-2 at a dose of 18 MIU/m(2) over 6 hours, followed by 18 MIU/m(2) over 12 hours, then 18 MIU/m(2) over 24 hours, and finally 4.5 MIU/m(2)/day for 3 consecutive days. rIFN-alpha-2a (10 MIU/m(2)) was given subcutaneously on Days 1-5. Courses were repeated every 4 weeks. Tumor sites were measured every 8 weeks. Toxicity was recorded using National Cancer Institute Common Toxicity Criteria. RESULTS: No major objective responses were noted. The median number of courses given was two. The median time to progression was 2 months and the median survival was 6 months (range, 2-25 months). However, 2 patients with melanoma involving >/= 2 visceral organs (1 with a high baseline serum lactate dehydrogenase level) and a third with soft tissue metastases achieved durable control of disease and were alive a median of 30+ months later. A fourth patient had a palliative response with reversal of melanosis and a survival of 7 months. This regimen was well tolerated and resulted in no serious long term adverse effects. CONCLUSIONS: The response rate for this regimen was no greater than 10% with Type I and II errors each not exceeding 10%. Nevertheless, occasional durable control of disease and the nonoverlapping toxicity profile with prior chemotherapy support consideration of this regimen in these patients who have limited second-line treatment options.

Melanoma metastatic to the spine: a review of 133 cases.

Although spine metastasis from melanoma is an uncommon event, it can pose a complex management problem. The presentation and natural history of melanoma metastatic to the spine has not been described in the medical literature. We have conducted a review of the records of 133 patients with melanoma metastatic to the spine in order to obtain retrospective data on demographic information, clinical presentation, disease course and survival. Patients with cutaneous, ocular and mucosal melanoma were all represented, but those with primary cutaneous tumours of the trunk were more prevalent than expected. Other sites of metastatic disease were present in nearly all patients and metastases to other skeletal sites were not unusual. Pain was the most common presenting symptom. The radiographic diagnosis was generally made easily by plain radiographs, computed tomography or magnetic resonance imaging, with the most frequent finding being a destructive lesion. Bone scan gave false-negative results 15% of the time. The median survival for the group was 4 months. It is concluded that melanoma metastatic to the spine represents a late event in the evolution of this illness. Palliation should be the goal of treatment, but symptom management should be individualized, bearing in mind the short anticipated survival of these patients.

Phase II trial of doxil for patients with metastatic melanoma refractory to frontline therapy.

Treatment options for patients with stage IV melanoma are limited. Based on differences in the toxicity and activity profiles of pegylated liposomal doxorubicin (doxil) compared to standard doxorubicin, we have conducted a phase II trial of doxil for patients with metastatic melanoma. Doxil was administered as a 60-90 min intravenous infusion every 21 days. The starting dose was 60 mg/m2 for the initial nine patients, but was subsequently reduced to 50 mg/m2 for the remainder due to toxicity issues. Thirty-two patients were enrolled in the trial. Ninety-one percent had received prior systemic therapy. There were no complete responses and two partial responses for an overall response rate of 6%. The dominant side effects included hand-foot syndrome, rash (occasionally severe), and stomatitis, consistent with reports from other trials using similar doses and schedules. We conclude that doxil does not demonstrate sufficient activity in metastatic melanoma to warrant further investigation into its use in this setting.

Coincident renal cell carcinoma and nonHodgkin's lymphoma: the M. D. Anderson experience and review of the literature.

PURPOSE: We recently observed nonHodgkin's lymphoma and renal cell carcinoma occurring simultaneously in several patients. We determine whether the incidence of co-occurrence of the 2 malignancies is greater than expected based on the incidence of each disease in the general population. MATERIALS AND METHODS: Patients diagnosed with simultaneous renal cell carcinoma and nonHodgkin's lymphoma were identified through the medical informatics data base at our cancer center. Charts of all patients were reviewed to collect detailed demographic information and confirm both diagnoses. United States population based cancer statistics (Surveillance, Epidemiology and End Results Program data) were used to arrive at the expected age adjusted incidence of co-occurrence of these diagnoses, and statistical analysis was performed to ascertain any differences between expected and observed incidences. RESULTS: We identified 41 cases with both diagnoses between 1954 and 1995, including 21 diagnosed after 1987. The latter group was used for statistical analysis. The observed rates of renal cell carcinoma developing in the nonHodgkin's lymphoma population (1.86) and nonHodgkin's lymphoma developing in the renal cell carcinoma population (2.67) were greater than expected and both reached statistical significance. CONCLUSIONS: There is a higher than expected incidence of co-occurrence of renal cell carcinoma and nonHodgkin's lymphoma. The cause remains speculative.

Phase II trial of 5-fluorouracil, interferon-alpha and continuous infusion interleukin-2 for patients with metastatic renal cell carcinoma.

BACKGROUND: This study was designed to evaluate the efficacy and toxicity of the combination of 5-fluorouracil, interferon-alpha, and interleukin-2 for patients with metastatic renal cell carcinoma. METHODS: Previously untreated patients with a Zubrod performance status of < or =2; adequate cardiac, pulmonary, and renal function; and absence of brain metastases were eligible. One course of therapy was 28 days. 5-fluorouracil was administered at a dose of 600 mg/m2/day as a continuous infusions on Days 1-5. Interleukin-2 also was administered as a continuous infusion on Days 1-5 at a dose of 2 million Roche U/m2/day. Interferon-alpha was given as a daily subcutaneous injection of 4 million U/m2/day. RESULTS: Fifty-five patients were enrolled in the trial and 52 were evaluable for response. All patients experienced fever and flu-like symptoms. Grade 3 or 4 nonhematologic toxic effects included hypertension (48%), dermatitis (12%), stomatitis (11%), and altered mental status (9%). There was one toxic death. Four complete responses and 12 partial responses were observed for a total response rate of 31% (95% confidence interval, 18-46%). The survival of responding patients was significantly better than that of nonresponding patients. The improvement in survival was even more significant when comparing patients with at least stable disease with those who progressed through treatment. CONCLUSIONS: The three-drug combination described in this study demonstrates activity. However, it appears to be more toxic than other regimens with similar response rates and cannot be recommended for standard practice. Changing the interleukin-2 route to subcutaneous administration may permit more continuous administration with less toxic effects.

Phase II trial of alternating weekly chemohormonal therapy for patients with androgen-independent prostate cancer.

Two distinct regimens of weekly chemotherapy for hormone-refractory prostate cancer were combined in an alternating schedule and tested in a Phase II trial to determine efficacy and toxic effects. Forty-six patients with hormone-refractory prostate cancer and rising prostate-specific antigen (PSA) levels entered the trial. Therapy consisted of doxorubicin (20 mg/m2/week) plus oral ketoconazole (400 mg three times a day) given at weeks 1, 3, and 5 and vinblastine (5 mg/m2/week) plus oral estramustine (140 mg three times a day) given at weeks 2, 4, and 6. No therapy was given at weeks 7 and 8. Replacement doses of hydrocortisone were administered throughout treatment to counteract potential adrenal insufficiency secondary to the ketoconazole. In 67% of patients (31 of 46), the PSA declined by 50% or greater for a minimum duration of 8 weeks (95% confidence interval, 52-80%). Among the 16 patients with measurable soft tissue disease, there were 12 responses (75%; 95% confidence interval, 47-92%). The median duration of response was 8. 4 months (1.8-14.9). The median survival for the entire group was 19 months. The median survival of PSA responders has not been reached, whereas that of nonresponders was 13 months (P = 0.010). Seventy-six percent of symptomatic patients noted improvement. Hematological toxicity was modest and was managed without growth factors. Peripheral edema (49%) and deep venous thrombosis (18%) were the most common nonhematological toxicities. The alternating weekly regimen of chemohormonal therapy is active for hormone-refractory prostate cancer, providing a high rate of symptom control, soft tissue response, and PSA decline.

Intraabdominal desmoplastic small cell tumor. Report and discussion of five cases.

BACKGROUND: Intraabdominal desmoplastic small cell tumor is a rare malignancy that has only recently been described. In the adult population, it is usually seen in young males. The tumor appears to arise as a dominant mass or masses in the abdominal cavity, particularly in the pelvic region, without a clear visceral origin. Multiple small tumor nodules may be found attached to the peritoneal surface. Visceral metastases occur late in the clinical course, ultimately leading to the patient's death. Five young adults with intraabdominal desmoplastic small cell tumors were treated with aggressive chemotherapy. METHODS: Charts of five patients treated between 1990 and 1995 were reviewed. The diagnosis of desmoplastic small cell tumor was made on the basis of typical clinical and radiographic findings, light microscopy showing hyperchromatic cells surrounded by a desmoplastic stroma, and immunohistochemistry demonstrating markers of epithelial and mesenchymal differentiation. Patients were treated with aggressive combination chemotherapy consisting of alternating regimens similar to those used as testis cancer salvage therapy. RESULTS: Four of the five patients demonstrated a partial response to chemotherapy. Two of the responding patients had surgery with the intent of removing all residual disease, but this was successful in only one case. All patients have died from their cancer. CONCLUSIONS: Intraabdominal desmoplastic small cell tumors are sensitive to combination chemotherapy. It appears that treatment offers good palliation and may prolong survival in isolated cases. However, even the patients managed with the most aggressive medical and surgical approaches are not cured and ultimately die of their disease.

Escalated MVAC with or without recombinant human granulocyte-macrophage colony-stimulating factor for the initial treatment of advanced malignant urothelial tumors: results of a randomized trial.

PURPOSE: Hematopoietic growth factors have been shown to ameliorate the side effects of chemotherapy. Here we assess the ability of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) to increase the dose-intensity and reduce the side effects of escalated methotrexate, vinblastine, doxorubicin, and cisplatin (MVAC) chemotherapy. PATIENTS AND METHODS: A prospective randomized trial to compare escalated MVAC versus escalated MVAC with rhGM-CSF was conducted. All patients were treated at The University of Texas M.D. Anderson Cancer Center (UTMDACC) and had a metastatic or unresectable urothelial tumor. Forty-eight patients were randomized (25 to MVAC with rhGM-CSF and 23 to escalated MVAC alone). The clinical characteristics of the study populations were similar (ie, degree of tumor dissemination and performance status). RESULTS: The dose-intensity in the two arms of the study did not differ significantly. No difference in the frequency of bacteriologically documented infections occurred between the two study arms. CONCLUSION: The use of the hematopoietic growth factor rhGM-CSF did not result in an increased dose-intensity of escalated MVAC. The inability to increase the dose-intensity of MVAC further was a result of nonhematologic side effects of the chemotherapy. Escalation of treatment delivered at its median-tolerated dose is unlikely to result in additional therapeutic benefit for patients with common solid tumors. Future development of therapy may require the development of new agents or concepts, rather than modification of existing therapies.

A phase I study of FAP (5-Fluorouracil, α-interferon and cisplatin) combined with methotrexate for the treatment of advanced urothelial malignancies.

Combination chemotherapy using 5-fluorouracil and α-interferon is active against advanced urothelial cancers. Its toxcity profile appears favorable such that addition of other active agents (i.e., cisplatin and methotrexate) may improve its therapeutic window. Our goal was to identify the starting dose of FAP (5-fluorouracil-α-interferon-cisplatin) that will permit delivery of methotrexate in patients with advanced chemotherapy-refractory urothelial cancers. This is a phase 1 study in which the dose level of FAP that permitted delivery of two doses of methotrexate in 28 days will be considered the maximum tolerated dose (MTD). This is based on experiences with MVAC chemotherapy in which only half of the patients received two doses of methotrexate in a 28 days cycle. The dose level identified as the MTD is as follows: 5-FU 500 mg/m(2) by intravenous continuous infusion daily for 5 days in weeks 1 and 4; α-interferon 5 MU/m(2) subcutaneously daily for 5 days simultaneously with 5-FU infusion in weeks 1 and 4 and thrice weekly during weeks 2 and 3; and cisplatin 25 mg/m(2) plus methotrexate 30 mg/m(2) intravenously once weekly for 4 weeks. The treatment will be repeated every 6 weeks. Most of the significant toxic effects, including mucositis, neutropenia, and thrombocytopenia, are encountered during weeks 3 and 4. This study showed that the MTD for FAP combined with methotrexate has been determined for phase II studies. Exploration of alterative regimen such as FAP in the treatment of advanced urothelial cancer is warranted. FAP has a unique mechanism of action and acceptable toxicity profile that may allow novel drug combinations and improved therapeutic efficacy.

Phase II trial of low dose gamma-interferon in metastatic renal cell carcinoma.

We conducted a phase II trial to confirm the activity of fixed, low dose gamma-interferon in metastatic renal cell carcinoma. A total of 35 patients with metastatic renal cell carcinoma, who had not received prior immunotherapy and who had a Zubrod performance status of 2 or less, was enrolled in this study. Primary tumors were controlled by nephrectomy or embolization before treatment began. gamma-Interferon was administered weekly as a subcutaneous injection at a fixed dose of 100 micrograms. Toxic effects were limited to low grade fever, chills and myalgias within 24 hours of injection. There were no incidences of grade 3 or 4 toxicity. Responses could be evaluated in 34 patients. There were 1 complete and 4 partial responses, for an objective response rate of 15% (95% confidence interval 5 to 32%). Durations of response to date are 21+, 17+, 13+, 9 and 2 months. We conclude that gamma-interferon is an active agent for metastatic renal cell carcinoma when administered according to this dose and schedule. The response rate compares favorably with those of alpha-interferon and interleukin-2, and toxicity is minimal. gamma-Interferon has excellent potential for use in combination with other biological or chemotherapeutic agents and in the adjuvant setting.

2'-deoxy-5-azacytidine increases binding of cisplatin to DNA by a mechanism independent of DNA hypomethylation.

The chemotherapeutic agents 2'-deoxy-5-azacytidine (DAC) and cisplatin (cDDP) have been shown in vitro to be synergystic in their cytotoxicity toward human tumour cells. We have investigated possible molecular mechanisms underlying this synergy using the plasmid pSVE3 in vitro and after transfection into CMT3 cells. Increased binding of cDDP to DAC-substituted DNA generated in vivo was confirmed by flameless atomic absorption spectrophotometry (FAAS). The plasmid used in these experiments was unmethylated suggesting that DAC was effective in enhancing cDDP binding to DNA without acting as a hypomethylating agent, but by directly changing the topology of DNA. The role of DNA methylation in cDDP binding was studied using methylated and unmethylated plasmid incubated in vitro with cDDP. Restriction analyses and FAAS measurement of bound platinum indicated that methylated DNA bound more cDDP than unmethylated DNA. In addition, in vivo studies confirmed the in vitro observations since replication of methylated plasmid was inhibited to a greater extent than unmethylated plasmid.

Protamine antibody production in diabetic subjects treated with NPH insulin.

Treatment with neutral protamine Hagedorn (NPH) insulin predisposes individuals with diabetes to anaphylactoid reactions when given bolus protamine for heparin reversal during cardiovascular procedures. To prospectively examine production of protamine antibodies, 30 patients with non-insulin dependent diabetes were followed for 12 months from initiation of therapy with porcine NPH or Lente insulin. Twenty-one subjects were randomly assigned to NPH (protamine containing) and nine controls to Lente (protamine free) insulin. Protamine specific IgG antibody was produced by 6/21 (29%) of NPH-treated subjects and 0/9 controls. Among NPH treated subjects, there was no difference between protamine antibody producers and non-producers with regard to age, race, weight, or pre-treatment glycosylated hemoglobin. Both producer and non-producer groups received similar amounts of insulin and protamine and achieved similar glycemic control. Insulin antibodies were made by 4/6 (67%) of protamine antibody producers and by 6/15 (40%) of non-producers (NS). The authors conclude that one of three new diabetics who are treated with porcine NPH insulin will make IgG protamine antibodies. These antibodies do not affect insulin requirements, glycemic control, or insulin antibody production. Because of the frequency of protamine antibody production and the risk of anaphylaxis to bolus protamine administration in NPH treated diabetics, the authors suggest that NPH insulin-treated individuals should avoid heparin reversal by protamine.

Factors affecting the insulin autoantibody ELISA.

IgG antibodies to insulin are present in insulin-treated patients and are detected in the prodrome of untreated type I diabetes. Sporadic reports of autoantibodies to insulin suggest that they are also present in other disorders. To establish the incidence of insulin autoantibodies in other endocrine and autoimmune diseases an ELISA was used to examine sera from 529 subjects with no prior insulin therapy. These untreated patients included: normal controls (adults and children), newly-diagnosed type I diabetes, first-degree relatives of diabetics, type II diabetes, Graves' hyperthyroidism, and systemic lupus erythematosus. As a positive control group, 280 insulin-treated patients were studied. Measurement of IgG antibodies by direct binding to insulin coated plates was complicated by differences between adult and pediatric populations and by overlap of binding between treated and untreated subjects. Competitive inhibition with excess soluble human insulin overcame these problems and permitted identification of insulin specific binding. Using this approach insulin antibodies were most frequent in insulin-treated diabetics (98%) and in type I diabetics (37%) prior to treatment. The absolute numbers of subjects with insulin autoantibody in the other groups differed depending upon whether a cut-off for binding (mean + 2SD of controls) or for insulin inhibition of binding (45%) was used. Regardless of the criteria used there were subjects (2-24%) in all groups tested with circulating insulin-specific IgG autoantibody detected by ELISA. These low level antibodies detected in solid phase assays may be part of the normal immune repertoire.