Canine mast cell tumors: correlation of apoptosis and proliferation markers with prognosis

The Patnaik histologic grading system is commonly used to predict the behavior of cutaneous mast cell tumors (MCTs) in dogs, but it is less useful for grade 2 MCTs because they exhibit considerable variation in biological behavior. In this retrospective study, immunohistochemical staining for Ki-67, proliferating cell nuclear antigen (PCNA), and survivin and a standardized argyrophilic staining of nucleolar organizer regions (AgNOR) protocol were performed on 121 archived paraffin-embedded specimens of canine cutaneous MCTs, for which clinical follow-up data were available. Cox regression models indicated that the Ki-67 score (hazard ratio, 1.92; P < .001) and mean AgNOR score (hazard ratio, 2.57; P < .001) were significantly associated with Patnaik grade and survival time. A binary Ki-67 variable (cutoff point Ki-67 score = 1.8) was a significant predictor of survival for dogs with grade 2 MCTs. The estimated 1-, 2-, and 3-year survival probabilities for dogs with grade 2 MCTs and Ki-67 scores less than 1.8 were 0.92, 0.86, and 0.77, respectively (SEs, 0.08, 0.14, and 0.23, respectively; median not estimable). The corresponding survival probabilities for dogs with grade 2 MCTs and Ki-67 scores higher than 1.8 were 0.43, 0.21, and 0.21, respectively (SEs, 0.19, 0.18, and 0.18, respectively; median survival time, 395 days). No significant association was identified between survival and survivin score or PCNA score. This study shows that both mean AgNOR score and Ki-67 score are prognostic markers for canine MCTs. The Ki-67 score can be used to divide Patnaik grade 2 MCTs into 2 groups with markedly different expected survival times.     

Correlation of histologic grading of canine mast cell tumors with Ki67/PCNA/AgNOR/c‐Kit scores: 38 cases (2002–2003)

Introduction: The Patnaik grading system for canine cutaneous MCT is currently one of the best determinants of prognosis; however, clinical outcome does not always correlate with histologic grade. The development of molecular markers offers a potential advantage and may complement subjective grading. The primary purpose of this study was to correlate histologic grading to Ki67/PCNA/AgNOR/c‐Kit scores. Methods: Thirty‐eight dogs with cutaneous MCT underwent surgical resection. Tumors were graded, with expansion of grade II MCT to low, medium (or II only) and high. For statistical purposes, MCT grade I, II (low, medium, high) and III were assigned a score of 1, 2, 3, 4, or 5, respectively. Sections were processed for AgNOR staining and expression of PCNA, Ki67 and c‐Kit as previously described (modified biotin‐strepavidin with DAB substrate). Paraffin‐embedded canine tissue arrays were used as positive and negative controls (primary antibody replaced with pre‐immune sera). Parametric statistical testing was performed using Statview statistical software with P ≤ .05 as significant. Results: There were 12, 20, 5 and 1 grade II low, grade II medium, grade II high and grade III MCT, respectively. The mean Ki67 score was 9.114 (median 8.0, range 1–28), mean PCNA score was 26.25 (median 24.0, range 5–65), mean AgNOR score was 1.499 (median 1.35, range 1.02–2.76) and c‐Kit scores were +1 (9/37), +2 (19/37) and +3 (9/37). With parametric statistical testing, significantly positive correlations were found for Ki67/Grade, PCNA/Grade, AgNOR/Grade, Ki67/PCNA, Ki67/AgNOR and PCNA/AgNOR (all P < .0001). No significant correlation was found for c‐Kit and grade; however, +3 c‐Kit scores had statistically significantly higher grades than +2 c‐Kit scores (P = .0458). Conclusions: Ki67/PCNA/AgNOR scores are positively correlated to grade in dogs with MCT. Further studies to correlate Ki67/PCNA/AgNOR/c‐Kit scores with clinical variables are ongoing.     

Prognostic factors for treated canine malignant lymphoma.

The aim of this study was to investigate the prognostic importance of different clinical, immunohistologic and tumor proliferation characteristics in dogs with malignant lymphoma treated with chemotherapy. From 74 dogs with malignant lymphoma at least one enlarged peripheral lymph node was taken for biopsy before chemotherapy following a standardized protocol (vincristine, cyclophosphamide, prednisolone, doxorubicin, and L-asparaginase). The variables evaluated as prognostic factors were age, sex, and tumor stage, as well as histomorphologic grade (Kiel classification, Working Formulation), immunophenotype (using markers for CD3 and CD79a), and cell proliferation (Ki-67, proliferation cell nuclear antigen, mitotic index, and argyrophil nucleolar organizer regions [AgNORs]) in extirpated lymph nodes. All markers were used on routinely formalin-fixed, paraffin-embedded tissues. The AgNORs were assessed qualitatively, based on the AgNOR pattern distribution, and quantitatively using image analysis and routine counting. In both univariate and multivariate survival analyses, AgNORs were a valuable prognostic marker for the treatment of canine malignant lymphomas. Based on the results of the multivariate analysis longer survival time correlated with a B-cell type, a larger mean AgNOR area, a larger total AgNOR area, a shorter distance between two AgNORs, and a smaller AgNOR area to nucleus ratio. Longer disease-free survival time correlated with a smaller number of AgNORs per nucleus, a larger mean AgNOR area, a larger maximal AgNOR area, and a larger total AgNOR area. This study clearly demonstrates the additional benefit of the use of AgNORs in predicting treatment outcome in dogs with malignant lymphoma.     

Recurrence rate, clinical outcome, and cellular proliferation indices as prognostic indicators after incomplete surgical excision of cutaneous grade II mast cell tumors: 28 dogs (1994-2002)

The objectives of this study were to determine local recurrence rate, clinical outcome, and prognostic value of the number of argyrophylic nucleolar organizer regions (AgNORs), presence of proliferating cell nuclear antigen (PCNA), and number of Ki-67-positive nuclei after incomplete surgical excision of canine cutaneous grade II mast cell tumors (MCTs). This retrospective study included 30 MCTs in 28 dogs. Medical records were examined and follow-up information was obtained from owners and referring veterinarians. Only cases in which excision was incomplete and no anvcillary therapy (other than prednisone) for MCT was given were included. Paraffin-embedded tumor tissues were retrieved for AgNORs, PCNA, and Ki-67 staining. Median follow-up time was 811.5 days. Seven (23.3%) tumors recurred locally. Median time to local recurrence was not reached with a mean of 1,713 days. The estimated proportions of tumors that recurred locally at 1, 2, and 5 years were 17.3, 22.1, and 33.3%, respectively. Eleven (39.3%) dogs developed MCTs at other cutaneous locations. Median progression-free survival was 1,044 days. Median overall survival was 1,426 days. The combination of Ki-67 and PCNA scores was prognostic for local recurrence (P = .03) and development of local recurrence was prognostic for decreased overall survival (P = .04). Results suggest that a minority of incompletely excised MCTs recur. Therefore, ancillary local therapies may not always be necessary. However, local recurrence can negatively affect survival of the affected dogs. Cellular proliferation indices may indicate the likelihood of MCT recurrence after incomplete excision.     

Outcomes and prognostic variables associated with primary abdominal visceral soft tissue sarcomas in dogs: A Veterinary Society of Surgical Oncology retrospective study

Primary abdominal visceral soft tissue sarcomas (STSs) are rare tumours in dogs with little information available on outcomes. The goal of this retrospective, multi‐institutional study was to describe the common tumour types, location and prognostic factors associated with primary abdominal visceral STSs. Medical records were searched for dogs with primary abdominal visceral STSs at six institutions and were retrospectively reviewed. Tumours were graded using the previously described grading scheme for STSs of the skin and subcutis when information in the histopathology report contained adequate details. Forty‐two dogs were included in the study. Five dogs had grade I tumours, 11 had grade II and 15 had grade III tumours. The most common tumour type was leiomyosarcoma (38.1%). The most common tumour locations were the spleen (47.6%) and small intestine (23.8%). The local recurrence rate was low (4.7%). Metastasis was present at the time of surgery in 23.8%, and the overall metastatic rate was 40.4%. Mitotic index of ≥9 was associated with significantly shorter survival time (MST 269 days) compared with a mitotic index of <9 (MST not reached). The MST for grade I STSs was not reached, was 589 days for grade II and 158 days for grade III. Dogs with grade III tumours were more likely to develop metastatic disease. Neither location of the primary tumour nor the histologic subtype was associated with survival time. Histologic grading of abdominal visceral STSs using the previously described scheme is prognostic and should be provided on histopathology reports.     

Prognostic significance of intratumoral microvessel density in canine soft-tissue sarcomas

The prognosis of canine soft-tissue sarcomas (STS) has traditionally been based on histologic grading. We have recently demonstrated the prognostic value of cellular proliferation markers in canine STS. Another method of predicting the behavior of neoplasms is intratumoral microvessel density (IMD), which is a measure of tumor angiogenesis. The prognostic significance of IMD has been documented in many human neoplasms and in a limited number of canine and feline neoplasms. To evaluate the prognostic value of IMD in canine STS, we studied 57 STS and compared IMD with histologic features, histologic grade, cellular proliferation, metastatic propensity, and survival. Using immunohistochemistry, the STS were labeled with anti-factor VIII-related antigen (FVIII-RA) and anti-CD31 antibodies to determine 3 IMD parameters: mean microvessel density, high microvessel density, and microvessel area. Using FVIII-RA and CD31, increasing IMD was statistically associated with increasing histologic grade, necrosis scores, and mitotic scores. Higher FVIII-RA IMD values were significantly associated with higher median argyrophilic nucleolar organizing region (AgNOR) values (as previously investigated) and increased metastatic propensity. Fibrosarcomas appear to be the least vascularized of STS. There is no correlation between IMD and survival. Our results indicate that IMD is of prognostic value for histologic grade, histologic features, cellular proliferation (based on AgNOR), and metastatic propensity of canine STS, specifically when using FVIII-RA as the endothelial marker. Assessing histologic grading, cellular proliferation, and IMD of canine STS at the time of diagnosis could therefore provide better prognostic information for the veterinary clinician.     

Argyrophilic nucleolar organizing regions and Ki67 equally reflect proliferation in fine needle aspirates of normal, hyperplastic, inflamed, and neoplastic canine lymph nodes (n = 101)

BACKGROUND: The count of argyrophilic nucleolar organizing regions (AgNOR) has been considered a useful variable that reflects cellular proliferation in canine lymph nodes, but it has not been compared with other markers of proliferation. Hypothesis: Ki67 and AgNORs are equally useful as markers of tissue proliferation in fine needle aspirates of canine lymph nodes. ANIMALS: A total of 101 dogs. MATERIAL AND METHODS: Prospective, observational study of a convenience sample of dogs. Two smears were prepared for a May-Gruenwald-Giemsa stain and a Ki67/AgNOR double stain. In addition, CD3/CD79a immunostaining was performed when cytologic examination revealed a lymphoma. The dogs were grouped as normal (n = 26), reactive hyperplasia (n = 25), lymphadenitis (n = 31), and lymphoma (n = 19), based on the physical examination and the cytologic findings. The AgNOR count/cell, AgNOR area/cell and the percentage of cells staining positive for Ki67 were evaluated in 100-167 cells (median, 113 cells) by using automatic image analysis. RESULTS: Mean (SD) AgNOR counts/cell were 1.36 +/- 0.19 in normal dogs, 1.55 +/- 0.26 in lymphadenitis, 1.65 +/- 0.32 in reactive hyperplasia, and 3.67 +/- 1.08 in lymphoma. The percentage of Ki67 positive cells was 2.67 +/- 0.99% in normal lymph nodes, 5.04 +/- 3.34% in lymphadenitis, 5.36 +/- 2.14% in reactive hyperplasia, and 30.2 +/- 10.8% in lymphoma. All variables were significantly higher in dogs with lymphoma compared with the other groups (P < .0001). The sensitivity and the specificity of the AgNOR count for diagnosing lymphoma were 95 and 96% at a cutoff value of >2.04 AgNORs/cell. The cutoff value for the Ki67 positive cells was >10.40% (sensitivity, 95%; specificity, 98%). CONCLUSION AND CLINICAL IMPORTANCE: The results indicated that both AgNOR and Ki67 counts were good diagnostic tools for assessment of proliferation in aspirates of canine lymph nodes.     

Cellular proliferation in canine cutaneous mast cell tumors: associations with c-KIT and its role in prognostication

Canine cutaneous mast cell tumor (MCT) is a common neoplastic disease in dogs. Due to the prevalence of canine MCTs and the variable biologic behavior of this disease, accurate prognostication and a thorough understanding of MCT biology are critical for the treatment of this disease. The goals of this study were to evaluate and compare the utility of the proliferation markers Ki67, proliferating cell nuclear antigen (PCNA), and argyrophilic nucleolar organizing region (AgNOR) as independent prognostic markers for canine MCTs and to evaluate the use of these markers in combination, as each marker assesses different aspects of cellular proliferation. An additional goal of this study was to evaluate the associations between cellular proliferation and c-KIT mutations and between cellular proliferation and aberrant KIT protein localization in canine MCTs. Fifty-six MCTs treated with surgical excision alone were included in this study. Each MCT was evaluated for Ki67 expression, PCNA expression, and KIT protein localization using immunohistochemistry; for AgNOR counts using histochemical staining; and for the presence of internal tandem duplication c-KIT mutations using polymerase chain reaction amplification. In this study, increased Ki67 and AgNOR counts were both associated with significantly decreased survival. On the basis of these results, we recommend that the evaluation of cellular proliferation, including evaluations of both Ki67 expression and AgNORs, should be routinely used in the prognostication of canine MCTs. Additionally, the results of this study show that MCTs with aberrant KIT protein localization or internal tandem duplication c-KIT mutations are associated with increased cellular proliferation, further suggesting a role for c-KIT in the progression of canine MCTs.     

Prognostic value of histologic stage and proliferative activity in canine malignant mammary tumors.

The aim of this study was to investigate the correlation between the histologic invasiveness (histologic stage) and various cell proliferation activity assays (quantity of argyrophil proteins associated with nucleolar organizer regions [AgNORs], mitotic activity, MIB1 [Ki67] immunohistochemical detection) for predicting the biologic behavior of malignant canine mammary tumors. Sixty specimens from malignant canine mammary tumors with no distant metastases (M0) at surgery were selected, and follow-up data were collected over a 2-year period. The histologic invasiveness was graded by histologic stage (stage 0 = tumors without stromal invasion; stage I = tumors with stromal invasion; stage II = tumors with neoplastic emboli in vessels), and the proliferative indices were expressed as MIB1 index (the percentage of nuclear area immunohistochemically stained by MIB1 antibody), mitotic index (the number of mitoses per 1,000 neoplastic cells), and AgNOR index (the ratio between mean AgNOR area of tumor cells and the mean AgNOR area of fibroblasts/lymphocytes). The measures of proliferative activity were compared among groups with different histologic stages, and the influence of different prognostic variables (histologic stage, AgNOR index, mitotic index, MIB1 index) on survival time was evaluated. A significant difference in the proliferation patterns was recorded between the different histologic stages for the mitotic index (P = 0.0006) and MIB1 index (0.0013). Among the different parameters considered, histologic stage (P < 0.05), AgNOR index (P = 0.0291), and MIB1 index (P = 0.014) revealed a significant association with prognosis in univariate analysis. AgNOR index for 1-year survival and histologic stage for 2-year survival were the most significant parameters influencing survival, as determined by multiple nonlinear logistic regression.     

Retrospective outcome evaluation for dogs with surgically excised,solitary Kiupel high‐grade,cutaneous mast cell tumours

Published outcomes for dogs with specifically high‐grade mast cell tumours (MCTs), controlled for clinical stage, are few. Clinical outcomes for 49 dogs with Kiupel high‐grade, clinical stage I, cutaneous MCTs were evaluated. Median survival time (MST) was 1046 days; 1 and 2‐year survival rates were 79.3% and 72.9%, respectively. At study end 24 dogs had died, 23 dogs were alive (median follow‐up 980 days) and 2 dogs were lost to follow‐up. Death was considered MCT‐related in 14 of 20 dogs with a known cause of death. Local tumour recurrence developed in nine dogs (18.4%); regional lymph node metastasis occurred in six dogs (12.2%); and a new MCT developed in 15 dogs (30.1%). Tumour location, histologic margin size and use of chemotherapy did not affect MST; increasing mitotic count (P = .001) and increasing tumour diameter (P = .024) were independently negatively prognostic. Six dogs that developed lymph node metastasis after surgery had worse MST (451 days) than 42 dogs that did not develop metastasis (1645 days); (P < .001). Our study suggests that dogs with local surgical control of clinical stage I histologically high Kiupel grade cutaneous MCT may have a long survival time; especially those with smaller tumours and a lower mitotic count. Our results suggest that evaluation of staging information and mitotic count may be equally helpful as histologic grading when making a prognosis; and highlight the importance of not relying on histologic grade alone when predicting survival for dogs with MCT.     

Immunohistochemical detection of proliferating cell nuclear antigen and Ki-67 in mast cell tumors from dogs

OBJECTIVE: To determine whether immunohistochemical detection of proliferating cell nuclear antigen (PCNA) and Ki-67 correlated with prognosis for dogs with cutaneous mast cell tumors (MCT). DESIGN: Case series. ANIMALS: 120 dogs with solitary cutaneous MCT that were excised. PROCEDURE: Information on signalment, history, and outcome was obtained by sending a questionnaire to referring veterinarians. Tumors were graded histologically, and immunohistochemical staining for Ki-67 and PCNA was performed. RESULTS: Survival rates 12, 18, and 24 months after surgery were significantly different among groups when dogs were grouped on the basis of histologic grade. Although mean number of PCNA-positive nuclei/1,000 tumor nuclei was significantly higher for dogs that died of MCT than for those that survived, there was great overlap in values. Mean number of Ki-67-positive nuclei/1,000 tumor nuclei was significantly higher for dogs that died of MCT than for those that survived, without any overlap in values between groups, and number of Ki-67-positive nuclei/1,000 tumor nuclei was significantly different among groups when tumors were grouped on the basis of histologic grades. For dogs with grade-II tumors, number of Ki-67-positive nuclei/1,000 tumor nuclei (< 93 vs > or = 93) was significantly associated with outcome (survived vs died). CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that for dogs with solitary cutaneous MCT, determining number of Ki-67-positive nuclei may be useful in predicting prognosis, particularly for dogs with grade-II tumors.     

Recurrence rates and clinical outcome for dogs with grade II mast cell tumours with a low AgNOR count and Ki67 index treated with surgery alone

Grade II mast cell tumours (MCT) are tumours with variable biologic behaviour. Multiple factors have been associated with outcome, including proliferation markers. The purpose of this study was to determine if extent of surgical excision affects recurrence rate in dogs with grade II MCT with low proliferation activity, determined by Ki67 and argyrophilic nucleolar organising regions (AgNOR). Eighty‐six dogs with cutaneous MCT were evaluated. All dogs had surgical excision of their MCT with a low Ki67 index and combined AgNORxKi67 (Ag67) values. Twenty‐three (27%) dogs developed local or distant recurrence during the median follow‐up time. Of these dogs, six (7%) had local recurrence, one had complete and five had incomplete histologic margins. This difference in recurrence rates between dogs with complete and incomplete histologic margins was not significant. On the basis of this study, ancillary therapy may not be necessary for patients with incompletely excised grade II MCT with low proliferation activity.     

Prognostic factors for cutaneous and subcutaneous soft tissue sarcomas in dogs

Soft tissue sarcomas (STSs) develop from mesenchymal cells of soft tissues, and they commonly occur in the skin and subcutis of the dog. Although phenotypically diverse with frequently controversial histogenesis, STSs are considered as a group because they have similar features microscopically and clinically. Following resection, local recurrence rates are low in general but vary according to histologic grade and completeness of surgical margins. Complete margins predict nonrecurrence. Even most grade I STSs with "close" margins will not recur, but propensity for recurrence increases with grade. The frequency of metastasis has not been accurately estimated, but it is believed to be rare for grade I STSs and most likely to occur with grade III STSs. However, metastasis does not necessarily equate with poor survival. High mitotic index is prognostic for reduced survival time. Further research is needed to determine more precise estimates for recurrence rates and survival as related to completeness of surgical margins and to delineate potential differences in metastatic rate and median survival time between grades. Other potential indicators of prognosis that presently require further investigation include histologic type, tumor dimension, location, invasiveness, stage, markers of cellular proliferation, and cytogenetic profiles. Common issues limiting prognostic factor evaluation include biases from retrospective studies, small sample sizes, poor verification of metastasis, inconsistent STS classification and use of nomenclature, difficulties in differentiating STS phenotype, and diversity of the study population (stage of disease and treatment status).     

Comparison of mitotic index and Ki67 index in the prognostication of canine cutaneous mast cell tumours

Proliferation markers are commonly used for prognostication of mast cell tumours. The aim of the study is to compare the relative abilities of Ki67 and mitotic index to predict survival in the same cohort of dogs with cutaneous MCTs. Histological grade, mitotic index and Ki67 index were performed in all samples and clinical information was obtained by a follow‐up questionnaire. Ninety‐five dogs were included in the study with a median follow‐up of 1145 days. Survival times varied significantly between categories of histological grade, mitotic index and Ki67 index. Multivariable analyses showed that the risk of dying due to MCT was similar in dogs with increased Ki67 index [hazard ratio, HR: 3.0 (95% CI 1.3–6.8)] or increased mitotic index [HR: 2.7 (95% CI 1.1–6.5)]. In conclusion, both mitotic index and Ki67 index were able to independently differentiate MCTs with worse prognosis. This distinction is particularly meaningful in selecting intermediate grade MCTs that may benefit from more aggressive local or systemic treatment.     

Prognostic Value of Argyrophilic Nucleolar Organizer Region (AgNOR) Staining in Feline Intestinal Lymphoma

Limited information is available on prognostic factors for cats with lymphoma. The quantity of argyrophilic nucleolar organizer region (AgNOR) proteins can be used as a measurement of cellular proliferative activity. To determine if AgNORs were of prognostic value for feline intestinal lymphoma, the silver staining technique was performed on paraffin-embedded sections of 31 cases. Mean number of AgNORs per nucleus ranged from 1.02 to 4.32. Twenty-four (78%) cats had small AgNORs and 7 (22%) had large AgNORs. All cats were treated identically with a combination chemotherapy protocol. Response to chemotherapy was 87%. Median remission duration and survival times were 120 days and 201 days, respectively. No significant correlation was found between mean number of AgNORs per nucleus or AgNOR size and remission rate, remission duration, or survival time. This study indicates that AgNOR staining is not a useful prognostic factor for cats with intestinal lymphoma.     

Apoptotic and proliferation indexes in canine lymphoma.

Proliferative and apoptotic fractions of tumors were evaluated in 41 dogs with lymphoma for prediction of response to chemotherapy. All dogs had advanced clinical stage tumors, were untreated prior to study, and received identical induction-remission chemotherapy. Tumor cell proliferation was determined in all pretreatment biopsy specimens and in 18 specimens collected at the time of clinical relapse from remission. Quantitative measures included mitotic index and immunoreactivities for proliferating cell nuclear antigen (PCNA) and Ki-67. Apoptotic index was evaluated from 40 dogs pretreatment and from 16 dogs at the time of first relapse. Pretreatment tumor values for Ki-67, PCNA, and apoptosis were compared with posttreatment values. The median first relapse-free interval (RFI) and overall survival (OS) time were 174 days and 445 days, respectively. Of the proliferation markers, only the results of the Ki-67 analysis were predictive for duration of the first RFI but not OS. Pretreatment apoptotic index was also predictive of the duration of first RFI but not OS. No significant predictive value for comparison of the pretreatment and postrelapse values was demonstrated. Ki-67 labeling index and apoptotic indexes were combined to form both a proliferation/apoptotic ratio (PAR) and a sum, or turnover index. Only the PAR was predictive for duration of first RFI on multivariate analysis. Other variables that were evaluated for their influence on treatment outcome included patient age, weight, gender, clinical stage, clinical substage, and tumor immunophenotype. Of these variables, only immunophenotype was found to be of value for predicting duration of first RFI and OS.     

Ki-67 and Vascular Endothelial Growth Factor Expression in Intracranial Meningiomas in Dogs

Background: Tumor proliferation in human intracranial meningiomas can be defined by the reactivity of the monoclonal antibody MIB-1 to the Ki-67 antigen. Vascular endothelial growth factor (VEGF), a pro-angiogenic factor, is a predictive marker for survival of dogs with intracranial meningiomas.
Hypothesis: Ki-67 is expressed in canine intracranial meningiomas and is associated with VEGF expression. Ki-67 expression is a prognostic marker for patient outcome.
Animals: Seventy client-owned dogs with WHO grade I intracranial meningiomas.
Methods: Retrospective study assessing the degree of immunostaining for Ki-67 by MIB-1 and VEGF expression in intracranial meningioma tissue from dogs. MIB-1 Labeling Index (LI) was calculated with Image J NIH-software. Extent, intensity, and distribution of VEGF-expression was assessed semiquantitatively. Cross tabulations with Fisher's exact tests and nonparametric Spearman's rank correlations were performed to identify associations between VEGF expression and MIB-1 LI. Fifteen dogs underwent postsurgical radiotherapy and were included in survival analysis. The effect of MIB-1 LI on survival was examined by Kaplan-Meier and Cox proportional hazards regression procedures.
Results: Ki-67 staining was positive in 91% (64/70) and VEGF expression was detected in 96% (67/70). There was no significant association between VEGF expression and MIB-1 LI. MIB-1 LI was not associated with survival.
Conclusions and Clinical Importance: MIB-1 antibody can be used to document cell proliferation in intracranial meningiomas in dogs, but does not predict outcome. No association between VEGF as a marker of angiogenesis and tumor proliferation was found. Angiogenesis might be a more important predictor of meningioma activity in dogs than is Ki-67.     

Canine subcutaneous mast cell tumors: cellular proliferation and KIT expression as prognostic indices

Molecular assays are widely used to prognosticate canine cutaneous mast cell tumors (MCT). There is limited information about these prognostic assays used on MCT that arise in the subcutis. The aims of this study were to evaluate the utility of KIT immunohistochemical labeling pattern, c-KIT mutational status (presence of internal tandem duplications in exon 11), and proliferation markers--including mitotic index, Ki67, and argyrophilic nucleolar organizing regions (AgNOR)--as independent prognostic markers for local recurrence and/or metastasis in canine subcutaneous MCT. A case-control design was used to analyze 60 subcutaneous MCT from 60 dogs, consisting of 24 dogs with subsequent local recurrence and 12 dogs with metastasis, as compared to dogs matched by breed, age, and sex with subcutaneous MCT that did not experience these events. Mitotic index, Ki67, the combination of Ki67 and AgNOR, and KIT cellular localization pattern were significantly associated with local recurrence and metastasis, thereby demonstrating their prognostic value for subcutaneous MCT. No internal tandem duplication mutations were detected in exon 11 of c-KIT in any tumors. Because c-KIT mutations have been demonstrated in only 20 to 30% of cutaneous MCT and primarily in tumors of higher grade, the number of subcutaneous MCT analyzed in this study may be insufficient to draw conclusions on the role c-KIT mutations in these tumors.     

Proposal of a 2-tier histologic grading system for canine cutaneous mast cell tumors to more accurately predict biological behavior

Currently, prognostic and therapeutic determinations for canine cutaneous mast cell tumors (MCTs) are primarily based on histologic grade. However, the use of different grading systems by veterinary pathologists and institutional modifications make the prognostic value of histologic grading highly questionable. To evaluate the consistency of microscopic grading among veterinary pathologists and the prognostic significance of the Patnaik grading system, 95 cutaneous MCTs from 95 dogs were graded in a blinded study by 28 veterinary pathologists from 16 institutions. Concordance among veterinary pathologists was 75% for the diagnosis of grade 3 MCTs and less than 64% for the diagnosis of grade 1 and 2 MCTs. To improve concordance among pathologists and to provide better prognostic significance, a 2-tier histologic grading system was devised. The diagnosis of high-grade MCTs is based on the presence of any one of the following criteria: at least 7 mitotic figures in 10 high-power fields (hpf); at least 3 multinucleated (3 or more nuclei) cells in 10 hpf; at least 3 bizarre nuclei in 10 hpf; karyomegaly (ie, nuclear diameters of at least 10% of neoplastic cells vary by at least two-fold). Fields with the highest mitotic activity or with the highest degree of anisokaryosis were selected to assess the different parameters. According to the novel grading system, high-grade MCTs were significantly associated with shorter time to metastasis or new tumor development, and with shorter survival time. The median survival time was less than 4 months for high-grade MCTs but more than 2 years for low-grade MCTs.     

Cellular proliferation in tumors: a review of methods, interpretation, and clinical applications

Cellular proliferation with altered control is one of the 1st characteristics of a neoplastic cell population. Although tumor growth reflects both tumor cell replication and cell loss (opposing growth and antigrowth factors), most studies published in veterinary literature used immunohistochemistry (proliferating cell nuclear antigen [PCNA]; Ki-67 [MIB-1]) or staining for argyrophilic nucleolar organizer regions (AgNORs) to measure proliferation. These studies have appeared in the veterinary literature for more than a decade, describing associations between proliferation indices and histologic grade, biological behavior, and clinical outcome for some tumor types but no clinically relevant associations for other tumors. The results of these studies are summarized here. Methods for evaluation of the numerous regulatory proteins that direct the cell throughout its cycle are now available and should allow more precise identification of cellular aberrations in cancer.