Mast cell tumor (MCT) is a common neoplasm in dogs,

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1 J Vet Intern Med 2003;17: Relationship of Disease Progression and Plasma Histamine Concentrations in 11 Dogs with Mast Cell Tumors Taketo Ishiguro, Tsuyoshi Kadosawa, Satoshi Takagi, Gonhyung Kim, Tomohiro Ohsaki, Darko Bosnakovski, Masahiro Okumura, and Toru Fujinaga Plasma histamine concentrations (PHCs) were measured serially over 9 months or until death in 11 dogs with mast cell tumors (MCTs). Eight dogs had grossly visible disease and the other 3 dogs had microscopic disease. Initial PHCs in the dogs with gross disease were significantly higher than PHCs in healthy dogs (median, 0.73 ng/ml and 0.19 ng/ml respectively; P.009), whereas initial PHCs in dogs with microscopic disease showed no difference from controls. Seven dogs subsequently had progressive increases in PHC, and developed hyperhistaminemia (median, 14.0 ng/ml; range, ng/ml). These 7 dogs died from MCTs, and 1 had general weakness with rapid lysis of a large tumor burden after radiation therapy. PHCs of the other 4 dogs were less than 1 ng/ml during the study. These 4 dogs were still alive with adequate control of the tumor at the conclusion of the study. Four of the 11 dogs initially had gastrointestinal (GI) signs, which abated soon after administration of histamine-2 (H- 2) blockers. No significant difference was found between PHCs in dogs with GI signs and those without GI signs (median, 0.86 ng/ml and 0.35 ng/ml, respectively). Thereafter, 7 dogs had serious GI complications for which H-2 blocker therapy was ineffective. PHCs in these 7 dogs were extremely high (median, 12.2 ng/ml; range, ng/ml). Results of this study demonstrated that PHC was one factor related to disease progression, and indicated that marked hyperhistaminemia was associated with the GI signs refractory to H-2 blocker therapy in dogs with MCTs. Key words: Gastrointestinal signs; Hyperhistaminemia; Mastocytoma. Mast cell tumor (MCT) is a common neoplasm in dogs, accounting for 16 21% of all skin and subcutaneous tumors. 1 The clinical course of the disease varies from benign to highly malignant. 2 Histologic grade appears to be the most consistent parameter to predict the biologic behavior of MCT among a number of different parameters previously described. 3 6 Survival time based on grade after surgical excision has been reported in several studies. In 1 study, percentages of dogs surviving at least 1,500 days were 83% for grade I MCT, 44% for grade II MCT, and 6% for grade III MCT. 4 In another, 6 survival rate 12 months after surgery was 100% for grade I MCT, 71% for grade II MCT, and 24% for grade III MCT. Surgery and radiation therapy are the treatment options for localized MCT. 1,7 Chemotherapy may be indicated when surgery or radiation therapy is not feasible or as an adjunct to those treatments. 1 Clinical signs most commonly associated with MCT can be related directly to the cytoplasmic secretory granules that contain biologically active substances such as histamine, heparin, and proteolytic enzymes. 3 Disorders caused by release of the granule contents are gastroduodenal ulceration and perforation, delayed wound healing, hypotensive shock, local ulceration and swelling, and coagulation abnormalities. Gastroduodenal ulceration is a common problem, and progresses in some dogs to perforation and death. 3,8 Hyperhistaminemia is regarded as a main factor contrib- From the Laboratory of Veterinary Surgery, Department of Veterinary Clinical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan. Reprint requests: Taketo Ishiguro, DVM, Laboratory of Veterinary Surgery, Department of Veterinary Clinical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, , Japan; ishiguro@vetmed.hokudai.ac.jp. Submitted April 23, 2002; Revised August 5, 2002; Accepted October 21, Copyright 2003 by the American College of Veterinary Internal Medicine /03/ /$3.00/0 uting to gastroduodenal ulceration and perforation. 3,8,9 In 1 report, 20 (83%) of 24 dogs that died of progressive MCTs had gastroduodenal ulceration at postmortem examination. 8 Histamine release from MCTs may occur spontaneously in physiologically active tumors or release can be triggered by aggressive manipulation, chemotherapy, or radiation therapy Available information on plasma histamine concentrations (PHCs) in dogs with MCTs is limited. Fox et al 9 reported that PHCs in dogs with MCTs were significantly higher than those in healthy dogs and were not related to clinical stage of disease, histologic grade, or tumor size. The relationship between variations in PHC and the clinical course of MCTs has not been described. The purpose of this study was to describe variations of PHCs in dogs with MCTs and to evaluate the relationship between PHC and metastatic status, tumor volume, clinical signs, and survival time. In addition, the potential efficacy of PHC as a diagnostic tool and prognostic indicator in dogs with MCTs was evaluated. Materials and Methods Patient Selection In 11 dogs with histologically confirmed MCTs, PHCs were measured over 9 months or until death. All data were reviewed at the Veterinary Teaching Hospital, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan, from February 1997 to April Sample Preparation and Plasma Histamine Assay Blood samples for histamine measurement were mixed with ethylenediaminetetraacetic acid in plastic tubes a and immediately centrifuged at 800 g for 10 minutes at 4 C. Plasma was carefully separated from the upper portion of the sample more than 0.5 cm above the buffy coat to avoid contamination with leukocytes or tumor cells and finally frozen at 30 C until assay. All blood samples were obtained at least 2 days after surgery, irradiation, or administration of any chemotherapeutic agent except prednisolone. b Prednisolone, in most dogs, had been used daily for more than 7 days before subsequent blood sampling. PHC was measured by a commercial immunoassay

2 Plasma Histamine in Canine Mast Cell Tumor 195 recorded as an indicator of tumor-related complications. Physical examinations, CBC, and serum biochemistry periodically were performed on all dogs. Survival time was defined as the time interval between the date of initial PHC measurement and the date of death. Statistical Analysis Categorization was done as follows. Dogs with MCTs or healthy dogs, presence or absence of GI signs, survival time, and the relation to PHC were analyzed by Mann-Whitney U-test. The difference between initial and final PHC was tested by using Wilcoxon signed-ranks test. Pearson s correlation coefficient was used to evaluate the relationship between tumor volume and PHC. Range and median were reported. For statistical purpose, undetectable PHC was set at 0.11 ng/ ml, and microscopic disease that showed only histologic evidence of tumor cells extending to the margins of surgical excision was set at 0 cm 3. Statistical significance was established as P.05. Fig 1. Plasma histamine concentrations (PHCs) during the study period in 11 dogs with mast cell tumors (MCTs). (A) Open symbols represent PHCs in dogs that died from MCT. (B) Solid symbols represent PHCs in the dogs that were still alive with adequate control of the disease during this study period. kit c in Mitsubishi Kagaku Bio-Clinical Laboratories. 14 This assay has been validated for in vitro or in vivo use in the dog PHCs in dogs with MCTs were compared to those in control dogs (3 intact male and 2 intact female adult healthy Beagles). Patient Assessment Age, gender, breed, weight, and history of treatments were recorded for each dog. Tumor location was described as head and neck, trunk, extremity, or multiple. Histologic examination was performed at the Laboratory of Comparative Pathology, in Graduate School of Veterinary Medicine, Hokkaido University. Tumors were graded on the basis of the criteria reported by Patnaik et al. 4 Systemic spread of the disease was assessed by physical examination, peripheral blood or buffy coat smear, cytologic examination or biopsy of regional lymph node, radiography, abdominal ultrasonography (with cytologic examination of lesions if abnormalities were observed), and postmortem examination. In this study, determination of metastasis by cytologic examination was made when multiple clusters of mast cells were detected in specimens. Tumor volume was calculated with the formula L D 2 / 6, where L was the longest diameter and D was the diameter at right angles. 18 When multiple tumors or involved lymph nodes were present, their volumes were added to determine total tumor volume. Metastatic status was categorized into 4 levels: no metastasis, regional lymph node involvement, distant metastasis, and no additional metastasis. No metastasis was defined as no evidence of metastasis in dogs in which no metastatic lesions were previously found. No additional metastasis was defined as no new gross findings of metastasis after treatment in dogs in which metastasis previously was diagnosed. Gastrointestinal (GI) signs, including anorexia, vomiting, diarrhea, and melena, were Results Dogs with MCTs The median age of 11 dogs with MCTs was 10 years (range, 3 15 years). Of the dogs with MCT, 4 were intact males, 1 was a castrated male, 5 were intact females, and 1 was a spayed female. Breeds represented included mixedbreed dogs (n 5), Maltese (n 2), Pugs (n 2), a Cocker Spaniel (n 1), and a Shih Tzu (n 1). The median weight was 11.8 kg (range, kg). At the time of initial PHC measurement, 8 dogs had measurable tumors and 3 dogs had only microscopic disease in the incision site of previous surgery. Tumor location included the head and neck (n 2), trunk (n 1), extremities (n 5), and multiple (n 3). All tumors except an extracutaneous tumor in the oral cavity were grade II, and 1 tumor progressed from grade II to grade III at recurrence. Six dogs were treated with surgery. All dogs received chemotherapy consisting of prednisolone, doxorubicin, d prednisolone-vinblastine e combination, vincristine f -asparaginase g -prednisolone combination, or vincristine-cyclophosphamide h -prednisolone combination. Three dogs had more than 1 additional chemotherapeutic regimen. Nine dogs received orthovoltage radiation therapy. Ten dogs received cimetidine i (10 mg/kg IV or PO q12h) as a histamine-2 (H-2) blocker. Seven dogs died or were euthanized as a result of deterioration of their disease, and 4 dogs were still alive at the conclusion of the study. The overall median survival time was not reached, with a median follow-up of 248 days (range, 30 1,500 days). Plasma Histamine Concentrations The median frequency of PHC measurements in dogs with MCTs was 6 times (range, 4 13 times) and the median initial PHC was 0.39 ng/ml (range, ng/ml). The median PHC in the control group of 5 normal dogs was 0.19 ng/ml (range, ng/ml). No significant difference was found between initial PHCs in dogs with MCTs and concentrations in healthy dogs. When separating gross disease and microscopic disease, initial PHCs in dogs with gross disease (0.73 ng/ml; range, ng/ml) were significantly higher than PHC in normal dogs (P.009). Variations of PHC during the study period in dogs with MCTs are shown in Figure 1. Seven dogs that died from

3 196 Ishiguro et al MCTs showed progressive increases in PHC, except for temporary decreases after initial therapy, and finally had remarkable hyperhistaminemia. The median interval between the date of final PHC measurement and the date of death was 3 days (range, 0 36 days). In these 7 dogs, the median final PHC (14.0 ng/ml; range, ng/ml) was significantly higher than their median initial PHC (0.85 ng/ml; range, ng/ml) (P.003). PHCs of 4 dogs that were still alive remained stable, were not more than 1 ng/ ml throughout this study, and no differences were found between the initial and the final PHCs in these 4 dogs. Metastatic status in 11 dogs was no metastasis (n 4), regional lymph node involvement (n 6), and no additional metastasis (n 1) at the time of initial PHC measurement. The initial PHCs in dogs with lymph node involvement seemed to be higher than those in dogs with the other metastatic status. Progressive increases in PHC occurred in 7 dogs that finally had advanced metastatic status; 4 of these dogs developed distant metastasis and 3 dogs had extensive involvement of regional lymph nodes. Distant metastasis in 2 of the 4 dogs was confirmed by postmortem examination, and metastasis in the other 2 dogs was cytologically detected in aspirates from the liver or from intraabdominal lymph nodes. Four dogs with no metastasis and no additional metastasis showed little change in PHC during the study. Statistical analysis was limited because of the small number of samples. Median tumor volume in 11 dogs was 5 cm 3 (range, cm 3 ) at the time of initial PHC measurement. Tumor volumes in 9 dogs were determined at the time of final PHC measurement. Median tumor volume in these 9 dogs was 18 cm 3 (range, cm 3 ). The correlation between tumor volume and PHC was high (r.74) and statistically significant (P.007) at the time of initial measurement, but low (r.17) and not significant at the time of final measurement. Four of the 11 dogs had GI signs at the time of initial PHC measurement. The GI signs abated soon after administration of H-2 blockers in all dogs. No significant difference was found between PHCs in the dogs with GI signs and those without GI signs (median, 0.86 ng/ml and 0.35 ng/ml, respectively). Thereafter, 7 dogs showed GI signs terminally, for which H-2 blockers were ineffective. All of the signs could be attributed to the progression of MCT. One dog had general weakness with rapid lysis of a large tumor burden after initial irradiation. The median PHC in these 7 dogs that did not respond to H-2 blocker (12.2 ng/ ml; range, ng/ml) was approximately 64 times as high as the median PHC in the control group of healthy dogs and was significantly higher (P.002). The initial PHC in dogs with MCTs did not statistically predict survival longer than 9 months. Postmortem Examination Postmortem examinations were performed on 3 of the 7 dogs that died of MCTs; the other dogs were not examined at their owners request. One dog had metastatic lesions in the right submandibular and retropharyngeal lymph nodes, liver, spleen, and kidneys. Multiple ulcers and a perforation were observed in the stomach, and evidence was found of acute peritonitis. Another dog had metastatic lesions in the liver, spleen, kidneys, lungs, diaphragm, and internal thoracic wall. Multiple ulcers and erosions of the mucosa of stomach, duodenum, and cranial part of the jejunum were observed. A duodenal ulcer had perforated. In the 3rd dog, multiple ulcers in the mucosa of the esophagus caused by gastric reflux and erosions in the gastroduodenal mucosa were observed, and no obvious metastatic lesions were found in the thoracic and abdominal organs. Discussion In the present study, 7 of the 11 dogs showed progressive increases in PHC, except for temporary decreases after initial therapy, and finally had remarkable hyperhistaminemia. These 7 dogs had tumor development and died from the disease. The other 4 dogs that were still alive with adequate control of MCT showed little variations in PHC during the study period. This result indicated that PHC was 1 factor related to disease progression in the dogs with MCTs. It is presumed that histamine release from MCT can be triggered by various forms of therapy In this study, every dog received chemotherapy alone or chemotherapy in combination with surgery or radiation therapy or both. One dog experienced generalized weakness and anorexia, and had an extremely high PHC 2 days after initial irradiation. These findings appeared to be related to rapid lysis of the large tumor burden by therapy. In the other dogs, such complications of therapy were not observed. Histamine is rapidly metabolized, usually within 1 2 minutes, by histamine N-methyl-transferase and by diamine oxidase (histaminase). 19 If therapy-induced histamine release is acute, transient, and not remarkable, it is difficult to understand its influence on dogs with MCT. A positive correlation between tumor volume and PHC initially was observed in this study, whereas a previous study did not identify such a correlation. 9 The difference may have been a consequence of sample size and population bias in each study. The number of dogs in the present study was smaller and most of these dogs had grade II MCT. Finally, tumor volume in this study was not correlated to PHC. This result reflects metastatic ability of the tumor and intensity of the local therapy. In the 2 dogs that finally developed distant metastasis, progressive increases in PHC were observed in spite of volume reduction of measurable tumor after local therapy. This finding also may suggest that the degree of hyperhistaminemia cannot be estimated accurately by estimating detectable tumor volume in each dog under treatment. High PHCs reported in this study were observed in 7 dogs with tumor development; 4 of them had distant metastasis and the other 3 dogs had extensive lymph node involvement. Therefore, PHCs in dogs with MCTs appeared to be related to the tumor dissemination. Compared with a previous study, 9 no significant differences were found in PHCs among the different clinical stages. According to the World Health Organization (WHO), staging a tumor means to assess the extent of its local and systemic spread for dogs with MCT. 3 Recently, the suggestion has been made that this staging system may overestimate clinical stage of disease. 7,20,21 The guidelines of the WHO suggest that cytologic specimens

4 Plasma Histamine in Canine Mast Cell Tumor 197 need only be evaluated for the absence or the presence of mast cells. 22 However, mast cells also are found in cytologic specimens of lymph nodes, liver, spleen, and bone marrow from clinically healthy dogs. 7,20 Therefore, clinical stages in the previous study likely were overestimated. Histamine is a potent gastric secretagogue and evokes a copious secretion from parietal cells by acting on H-2 receptors. 23 Hyperacidity secondary to hyperhistaminemia is regarded as a main factor in gastroduodenal ulceration, which affects up to 80% of dogs with progressive MCT. 8 H-2 blockers such as cimetidine, ranitidine, and famotidine commonly are used to counteract the effect of histamine. In the present study, PHCs in dogs that responded to H-2 blockers were not significantly different from those without GI signs, whereas PHCs in dogs that did not respond to H-2 blockers were times higher than those in the control group of healthy dogs. Therefore, if GI signs that do not abate with H-2 blocker therapy are present in dogs with MCTs, the affected dogs may have marked hyperhistaminemia. The above observations also suggest that individual variation exists in the physiologic response to histamine, and dogs with GI signs despite H-2 blocker therapy may need more intensive support to block the effects of histamine. PHC at the time of initial measurement did not predict whether dogs with MCTs survived longer than 9 months. In this study, differences in therapeutic regimen and small sample size made it difficult to assess the effect of initial PHC on survival time. Most dogs that had a PHC higher than 1 ng/ml died within a few months after that measurement was obtained. Therefore, periodic measurements of PHC may provide additional information on prognosis. Recently, the suggestion has been made that PHC could be useful in assessing response to treatment and detection of micrometastatic disease. 9 Histamine is widely produced from not only neoplastic mast cells but also from many types of normal cells such as basophils, mast cells, bone marrow cells, macrophages, neutrophils, and vasoendothelial cells. Histamine release from these cells may be changed under various physiologic or pathologic conditions Therefore, if dogs with MCT had other diseases, especially allergic diseases, their influences on PHC should be taken into consideration. In this study, the degree of participation of histamine release from MCT to total PHC under 1 ng/ml was not clear. Further investigations on PHC in dogs with MCTs will be needed to define the usefulness of PHC as a diagnostic tool. 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