Mast cell tumors are the most common malignant skin cancer in dogs. There is substantial variability in the biological behavior of MCTs in dogs,1 and a histologic grading scheme developed for classification of MCTs affecting haired skin is one of the strongest prognostic indicators in affected dogs.2 However, histologic assessment is prone to operator subjectivity, with variable grading results when the same tumor is examined by different pathologists.3,4 Thus, many other prognostic factors, including various proliferative indices, DNA an-euploidy, c-kit staining pattern, microvessel density, and mitotic index, have been evaluated in an attempt to better predict the biological behavior of MCTs in dogs.5–26 All of these factors have been studied primarily in MCTs involving haired skin, and none have been validated for tumors arising in other anatomic locations.
Anatomic location has been described as a predictor of the biological behavior of MCTs in dogs, with studies27–32 reporting more aggressive behavior of MCTs involving gastrointestinal, digital, and visceral (hepatic, splenic, or both) locations. This aggressive behavior is manifested by increased systemic involvement of the MCT, including lymph node and distant metastasis; patients with aggressive MCTs frequently die of their disease. Little information exists on the behavior of mu-cosal or mucocutaneous MCTs, with most reports33–42 discussing only a limited number of cases involving these locations. In 1 study,43 MCTs located on the muzzle of 24 dogs had a high regional metastatic rate; however, the number of tumors involving mucous membranes versus haired skin was not specified. To our knowledge, there are no published studies of clinical outcome in dogs with oral or perioral MCTs or comparing outcome for dogs with primary MCTs involving mucous membranes versus haired skin.
Chemokines, chemotactic cytokines classically involved in leukocyte migration, and their receptors participate in tissue-specific metastasis and have been associated with survival times in human patients with a large variety of neoplasms.44–52 In people, there are approxi mately 50 chemokines identified to date,53 suggesting that multiple chemokine signaling pathways may act redundantly via overlapping chemoattractant properties. Although not solely responsible for lymphatic trafficking, CCR7 has been found to be largely involved in promoting lymph node metastasis in a number of cancers in humans.50–52 Similar studies54 in dogs are limited. The primary purpose of the study reported here was to determine clinical outcome for dogs with MCTs arising from the oral mucosa, oral mucocutaneous junction, or perioral region of the muzzle. Additionally, we wanted to ascertain whether CCR7 expression in the primary tumor was associated with regional lymph node metastasis or survival time.
Materials and Methods
Case selection criteria—Records of the Veterinary Diagnostic Laboratory at the University of Illinois at Ur-bana-Champaign and the Kansas State University Veterinary Medical Teaching Hospital from 1996 through 2006 were searched to identify dogs with MCTs arising from the oral mucosa, oral mucocutaneous junction, or perioral region of the muzzle. Dogs were eligible for inclusion in the study if the diagnosis had been confirmed by means of histologic or cytologic testing.
Medical records review—Medical records, if available, of dogs included in the study were reviewed for information on signalment, site of tumor origin, involvement of regional lymph nodes, treatment methods, and outcome. If the medical record was not available for review, the veterinarian who submitted the sample and the pet owner were contacted to obtain information. Results of standardized clinical staging were obtained, if performed, along with results of all routine diagnostic testing that was performed, including histologic evaluation of the primary tumor, cytologic evaluation of regional lymph node fine-needle aspirates, CBCs, and serum biochemical testing.
CCR7 immunohistochemical staining—Twenty of the 44 dogs in the study had formalin-fixed, paraffin-embedded tissue blocks of their MCTs available at the University of Illinois Veterinary Diagnostic Laboratory for immunohistochemical staining for CCR7; half of the 20 cases had documented lymph node metastasis. Tissue sections (5 μm thick) were prepared and deparaffinized in xylene and rehydrated in alcohol. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol for 15 minutes. Slides were then rinsed twice with wash buffer for 5 minutes each. Slides were placed in citrate buffer (pH, 6) with 500 mL of deionized water, and heat-induced epitope retrieval was performed in a pressure cooker.a Slides were held at 125°C for 30 seconds followed by 90°C for 10 seconds under 17 to 21 psi of pressure. The slides were removed and allowed to cool in citrate buffer for 2 minutes and then placed in wash buffer.b Nonspecific staining was minimized by blocking for 10 minutes with a buffered casein solution,c then blocking for 15 minutes with avidin and biotin.d Blocked slides were incubated with a rabbit monoclonal CCR7 primary antibodye at a 1:150 dilution for 30 minutes at room temperature and washed. Slides were then incubated with a biotinylated goat anti-rabbit im-munoglobulinf for 20 minutes at room temperature, washed, and then incubated with horseradish peroxi-dase-conjugated streptaviding for 20 minutes at room temperature and washed again. The reaction was developed with 3,3′-diaminobenzidine chromogen substrate bufferh for 5 minutes, and the slides were then coun-terstained with light toluidine blue and hematoxylin. Manufacturer-recommended positive controls, including mouse-derived spleen and lymph nodes, were used to optimize the CCR7 staining protocol. Primary CCR7 antibody cross-reactivity to canine tissues was validated via demonstration of expected and conserved staining patterns between normal mouse- and canine-derived lymph nodes. Normal canine lymph node was used as an internal standard positive control and was processed on the same slide as each individual MCT. Additional sections of each tumor and normal lymph node were similarly processed for use as negative controls with substitution of buffer solution for the primary antibody. The CCR7 labeling intensity of each tumor and the associated internal standard positive control was graded on a scale from 0 to 3, where 0 = no staining, 1 = light staining intensity, 2 = moderate staining intensity, and 3 = maximum staining intensity. A CCR7 staining score was then calculated as a ratio of staining intensity of the tumor to staining intensity of the internal standard positive control; potential scores ranged from 0 to 1.
Statistical analysis—For description of the biological behavior of MCTs, dogs were classified as either alive with no evidence of disease, dead as a result of the MCT, dead from unrelated causes, or lost to follow-up. Survival time was defined as the time from diagnosis to death or euthanasia because of progressive disease. Patients were censored from the survival analysis if they died of other causes, were lost to follow-up, or were still alive at the end of the study period. Descriptive statistics were used to describe the study population. Site of tumor origin and presence of lymph node metastases were evaluated for associations with survival time by use of the Kaplan-Meier product-limit method. The Wilcoxon Mann-Whitney test was used to test for an association between CCR7 staining scores and the presence of lymph node metastases. The KaplanMeier product-limit method was used to test for an association between dichotomized CCR7 staining scores (< 0.5 [low] vs ≥ 0.5 [high]) and survival time. The dogs in this subset were also censored as stated above. All analyses were performed with standard software.i Values of P < 0.05 were considered significant.
Results
Forty-four dogs with confirmed MCTs of the oral mucosa (n = 14), mucocutaneous junction (19), or perioral region of the muzzle (11) met the criteria for inclusion in the study. The diagnosis was confirmed by use of histologic testing in 42 dogs and cytologic testing in 2 dogs. Locations for the oral mucosal tumors included buccal mucosa (n = 5), gingiva (4), tongue (4), and sublingual region (1). Locations for the mucocutaneous tumors included the lower lips (n = 12), upper lips (6), and commissure (1). Tumors involving the perioral region of the muzzle were located close to the lip margin but did not involve it; locations of these tumors included the upper (n = 7) and lower (4) muzzle. The study population consisted of 32 spayed females, 10 neutered males, 1 sexually intact female, and 1 sexually intact male. Mean age at the time of diagnosis was 7.7 years (range, 1 to 14 years). There were 9 mixed-breed dogs, 5 Labrador Retrievers, 4 Cocker Spaniels, 2 Doberman Pinschers, 2 Golden Retrievers, 2 Miniature Poodles, 2 Boxers, and 18 additional dogs representing 18 breeds.
In 36 dogs, cytologic testing of the submandibular lymph nodes for evidence of regional lymph node metastasis had been performed at the time of diagnosis; in the remaining 8 dogs, testing for regional lymph node metastasis had not been performed. Twenty-six of the 44 (59%) dogs included in the study had documented submandibular lymph node metastasis at the time of diagnosis, representing 26 of the 36 (72%) dogs that underwent cytologic assessment of the submandibular lymph nodes. Frequency of lymph node metastasis was 7 of 14 (50%) for dogs with MCTs of the oral mucosa, 11 of 19 (58%) for dogs with MCTs of the mucocutaneous junction, and 8 of 11 (73%) for dogs with MCTs of the perioral region of the muzzle. Results of histologic evaluation of a regional lymph node were available for 6 dogs, and in all instances, results of histologic evaluation confirmed results of cytologic testing. This included 5 dogs with and 1 dog without regional lymph node metastasis.
Treatment of the dogs varied widely, with all dogs receiving some type of treatment, including surgery, chemotherapy, radiation therapy, intralesional corticoste-roid injections, or some combination of these modalities. Surgical excision was attempted in 33 dogs, and incisional biopsy was performed in 9 dogs. Two dogs had no surgical procedures or biopsy performed. Twenty dogs were treated with radiation therapy for curative intent (48- to 60-Gy total dose; n = 13) or palliation (8- to 36-Gy total dose; 7). Twenty-five dogs were treated with some form of chemotherapy. Drugs used included prednisone, chlorambucil, cy-clophosphamide, lomustine, and vinblastine, as single agents or in combination. Six dogs received intralesional triamcino-lone injections alone or in combination with other treatment. When grouped by number of treatment modalities used (ie, surgery, radiation, and chemotherapy), 15 dogs were treated with 1 modality (7 with surgery and 8 with chemotherapy), 14 dogs were treated with 2 modalities, and 15 dogs were treated with all 3 modalities. Numbers of dogs with mucosal, mucocutaneous, and perioral tumors were fairly evenly distributed among the treatment groups (Table 1). Subjectively, dogs with lymph node metastasis were more likely to have received all 3 modalities than were dogs without documented metastasis. Owing to the wide array of treatments, statistical analysis of treatment variables and their potential association with survival time was not performed.
Number of treatment modalities received in 44 dogs with MCTs.
No. of dogs (% of dogs in that subgroup) | |||||
---|---|---|---|---|---|
Variable | Surgery only | Chemo only | 2 modalities | 3 modalities | |
Location | Mucosal | 3 (21) | 4 (29) | 4 (29) | 3 (21) |
Mucocutaneous | 2 (10.5) | 2 (10.5) | 8 (42) | 7 (37) | |
Perioral | 2 (18) | 2 (18) | 2 (18) | 5 (46) | |
Lymph node status | Positive | 2 (8) | 6 (23) | 7 (27) | 11 (42) |
Negative | 3 (33) | 0 (0) | 5 (50) | 2 (20) | |
Not assessed | 2 (25) | 2 (25) | 2 (25) | 2 (25) |
Overall MST for all dogs was 52 months, with 1-, 2-, and 3-year survival rates of 68%, 54%, and 54%, respectively. Tumor location (oral mucosa, oral mucocutaneous junction, or perioral region of the muzzle) was not significantly associated with survival time (Figure 1). However, MST for dogs with lymph node metastasis (14 months) was significantly (P = 0.006) shorter than MST for dogs without documented lymph node metastasis (MST not reached; Figure 2). Twenty-one (48%) dogs died of mast cell disease, including 2 that died because of local disease, 15 that died because of local disease and metastasis, and 4 that died because of metastatic disease. Five of these dogs were confirmed by means of cytologic testing (n = 2) or necropsy (3) to have developed systemic mast cell disease involving distant lymph nodes, the spleen, and the liver. Twenty-three dogs were censored from the survival analysis; 3 dogs were lost to follow-up at 29, 40, and 53 months; 9 dogs died of other causes; and 11 dogs were still alive at the time of the study. Mean duration of follow-up for censored patients was 38.7 months.
For the 20 MCTs that underwent immunohisto-chemical staining, CCR7 staining intensity varied, with some tumors having intense expression (Figure 3) and other tumors having weak or no staining. The CCR7 staining scores were not significantly (P = 0.80) different between MCTs from dogs (n = 10) with regional lymph node metastasis and MCTs from dogs (10) without documented metastasis. Additionally, when CCR7 staining scores were dichotomized as low (< 0.5) or high (≥ 0.5), MST of dogs with low scores (52 months) was not significantly (P = 0.27) different from MST for dogs with high scores (6 months; Figure 4).
Discussion
Results of the present study suggested that in dogs with MCTs arising from the oral mucosa, oral mucocutaneous junction, or perioral region of the muzzle, the presence of regional lymph node metastasis at the time of diagnosis was a negative prognostic factor. However, prolonged survival times could be achieved, even in dogs with evidence of metastasis, with treatment. Additionally, CCR7 expression in the primary tumor was not significantly associated with the presence of regional lymph node metastasis or survival time.
In the present study, 26 of 36 (72%) dogs had cy-tologic evidence of regional lymph node metastasis at the time of diagnosis. In contrast, MCTs located on haired skin in other areas of the body reportedly have a much lower metastatic rate, with lymph node metastasis documented at diagnosis in ≤ 10% of affected dogs, depending on the study.1,55,56 Most dogs with MCTs can be expected to have a good outcome with appropriate treatment,1 with survival time affected by the treatments that owners elect to pursue. In previous studies,12,20,23,25 MSTs for dogs with aggressive MCTs ranged from 1.4 to 13 months. By contrast, in our study, dogs with perioral and oral MCTs had prolonged survival times with a median of 52 months, despite the high proportion with regional lymph node metastasis, with dogs with lymph node metastasis having an MST of 14 months. Although treatment regimens for dogs in the present study varied widely, multimodal treatment was used in most dogs.
Multiple factors have been evaluated in an attempt to predict how MCTs will behave in individual dogs. Many of the histologic features that have been shown to have some prognostic value, such as histologic grade, mitotic index, and proliferative indices, were studied only in MCTs of haired skin. Mast cell tumors involving mucosal surfaces have been anecdotally described as having a worse prognosis,1 but few reports describing MCTs in these locations have been published. On the basis of the limited published cases, clinical evidence would support a relatively benign behavior of MCTs arising from the eyelid margin or conjunctiva33,34,36-38,40 with a more aggressive behavior for MCTs involving the oral cavity. Two case reports35,39 describe dogs with MCTs of the lip that had local metastatic disease at the time of diagnosis, with survival times of < 6 months. In a report41 of 5 dogs with lingual MCTs, 2 had lymph node metastases, systemic metastases, or both at the time of diagnosis and 2 of the remaining 3 had local recurrence leading to euthanasia following surgical removal. In a study42 of 11 dogs with MCTs predominantly involving the mucous membranes of the genitalia or oral cavity, a significantly shorter MST was found when compared with 50 dogs with MCTs of haired skin. Finally, the muzzle location for MCTs was reported43 to have a biologically aggressive behavior characterized as a high rate of regional lymph node metastasis in 24 dogs; however, specific location and involvement of mucocutaneous junctions were not described. The present study provides further evidence that MCTs of the oral mucosa, oral mucocutaneous junction, or perioral region of the muzzle have an aggressive biological behavior.
The high rate of local regional lymph node metastases observed in dogs in the present study (59%) prompted an investigation of MCT CCR7 expression. In humans, overexpression of several chemokine receptors in various tumors has been correlated with clinical outcomes such as the presence of metastases and survival time.44–47,49,53,57–59 Specifically, expression of CCR7 by cancer cells has been associated with regional lymph node metastasis.48,50–52,60,61 However, in the present study, no relationships between CCR7 expression and nodal metastasis or survival time were identified. However, given the various chemokine receptors that may participate in cell migration, our results were not entirely surprising, in that early nodal metastasis of canine MCTs may not be solely attributed to CCR7 expression but may require the interaction of various chemoattractant stimuli. Perhaps evaluating a panel of a number of chemokine receptors would provide more meaningful information, and further study is suggested.
There were several limitations to the present study, with the most important being its retrospective nature. Information obtained was based on medical record information, referring veterinarian information, and owner recall. Staging tests were not uniformly performed in all cases and typically were limited to information on regional lymph node evaluation. Previous studies55,56,62 have found that results of abdominal ultrasonography and bone marrow aspiration are rarely positive when performing staging in dogs with systemic MCTs. Because few of the dogs in the present study died of systemic disease and many of the dogs had prolonged survival times, it appears unlikely that distant metastatic disease was present at the time of initial evaluation in many of these dogs. However, lymph nodes were not cytologically evaluated in all of the dogs, and it is possible that the true rate of regional nodal metastasis was higher than determined in this study. Owing to the substantial difference in MST between dogs with and without lymph node metastasis in the present study and findings in previous studies63,64 that lymph node size is not a reliable predictor of the presence or absence of metastasis, fine-needle aspiration with cytologic evaluation of the regional lymph nodes, whether enlarged or not, is recommended for all dogs with oral or perioral MCTs.
The lack of a standardized treatment protocol was another important limitation of the present study, but this was unavoidable given the variability in disease stage at the time of diagnosis, clinician bias and perception of prognosis, and the owners' desires and financial constraints. The wide range of treatments precluded statistical analysis of the various treatment protocols. Because survival time is almost certainly affected by treatment, it is possible that we would have identified differences in MSTs for dogs with MCTs in the 3 locations in the present study and in biological behavior of the tumors if all patients had been treated identically. However, because the 3 locations were fairly evenly represented among the treatment groups and the meta-static rate at the time of diagnosis and survival times for the 3 locations were similar, we would speculate that it is unlikely that MCTs at these 3 locations differed in their behavior. Similarly, survival times for dogs with and without lymph node metastasis may have been affected by the treatments received. However, a higher percentage of dogs with lymph node metastasis received trimodality treatment. The significantly worse MST for dogs with lymph node metastasis despite aggressive treatment suggested that metastatic disease in these dogs truly was a negative prognostic indicator.
An additional limitation of the present study was that only 20 archived tissue samples were available for CCR7 immunohistochemical analysis. Therefore, the power of this analysis was limited, and it is possible that evaluation of a greater number of cases would have identified a prognostic value for this test. Almost all tumors in people in which CCR7 staining has been reported to be prognostic for lymph node metastasis are carcinomas, including but not limited to primary tumors of the breast, stomach, pancreas, tonsil, lung, thyroid gland, and esophagus.45,46,48,51,52,60,61 However, we are not aware of any studies of CCR7 immunohis-tochemical analysis of round cell tumors. The CCR7 marker was chosen for evaluation in the present study because of its high association with lymph node metastasis, the most frequent metastatic site in this population of dogs with MCTs. Thus, although CCR7 was not found to be of prognostic value in dogs with MCT, the staining protocol for CCR7 was optimized in this study, which may be helpful for further evaluation of CCR7 in other cancers affecting dogs.
ABBREVIATIONS
CCR7 | Chemokine receptor type 7 |
MCT | Mast cell tumor |
MST | Median survival time |
Decloaking Chamber Plus, Biocare Medical, Concord, Calif.
Super Sensitive Wash Buffer, BioGenex, San Ramon, Calif.
Power Block Universal Blocking Reagent, BioGenex, San Ramon, Calif.
Avidin/Biotin Blocking System, Covance Research Products, Dedham, Mass.
CCR7 antibody [Y59] (ab32527), Abcam Inc, Cambridge, Mass.
Super Sensitive Rabbit Link, BioGenex, San Ramon, Calif.
Super Sensitive HRP Label, BioGenex, San Ramon, Calif.
Concentrated Liquid DAB Substrate Pack, BioGenex, San Ramon, Calif.
GraphPad Prism, version 3.03, GraphPad Software Inc, San Diego, Calif.
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