Immunohistochemical and clinical evaluation of p53 in canine cutaneous mast cell tumors

One hundred twenty-six cutaneous mast cell tumors obtained by excisional biopsy from 106 dogs were evaluated using immunohistochemical staining for the presence of p53 protein. A standard avidin-biotin immunohistochemical protocol was used incorporating a polyclonal antibody of rabbit origin (CM-1) as the primary antibody. Histopathologic grading of tumors was performed on hemotoxylin and eosin-stained samples. There was a significant difference in the percentage of cells staining positive for p53 for the histopathologic grades (P = 0.0005). Grade III tumors had a significantly greater p53 content than did grade I or II tumors (P < 0.05). Clinical data obtained retrospectively was available for 54 dogs. Tumor recurred in 19 of 54 (35.2%) dogs. Twenty-nine dogs died by the end of the study; 9 of 29 (31.0%) died of mast cell tumor disease. Histopathologic grade showed a significant negative association with survival time. Both clinical stage and histopathologic grade showed a significant negative association with time to recurrence. The percentage of cells staining positive for p53 did not significantly improve the forward analysis. Immunohistochemical detection of p53 did not appear useful in characterizing the clinical association between cutaneous mast cell tumor cellular features and survival time or time to tumor recurrence in dogs.     

Immunohistochemical expression of p27 and p21 in canine cutaneous mast cell tumors and histiocytomas

The objective of this study was to evaluate by immunohistochemical means the nuclear expression of p27 and p21 proteins in cutaneous mast cell tumors and histiocytomas of dogs. In mast cell tumors, nine of the 13 grade I tumors, 13 of the 19 grade II tumors, and 10 of the 15 grade III tumors showed no detectable or mild p27 immunoreactivity. In contrast, one of the 13 grade I tumors, 12 of the 19 grade II tumors, and 11 of the 15 grade III tumors showed moderate or marked p21 immunoreactivity. Nineteen of the 28 histiocytomas showed no detectable or mild p27 immunoreactivity, and 24 cases showed moderate or marked p21 immunoreactivity. These findings indicate that a loss or absence of p27 expression is an early pathogenic event in mast cell and histiocyte tumorigenesis and that p21 expression may be a marker of mast cell tumor progression and histiocytoma cell proliferation.     

Immunohistochemical evaluation of prostaglandin E2 and vascular endothelial growth factor in canine cutaneous mast cell tumours

Vascular endothelial growth factor (VEGF) and prostaglandin E2 (PGE-2) appear to play a critical role in tumour neovascularization. In this study, we have investigated the expression of VEGF and PGE-2 in 53 canine cutaneous mast cell tumours (MCTs). Immunohistochemistry of tissue sections revealed that VEGF and PGE-2 were expressed in all mast cell tumours studied. When the expression patterns of VEGF and PGE-2 were compared with tumour grade according to Patnaik criteria, the only significant correlation observed was between PGE-2 staining intensity and tumour pathological grade, with grade II and III tumours having higher PGE-2 staining, both in intensity and percentage of cells stained, than grade I tumours ( P < 0.05).     

Cytological grading of canine cutaneous mast cell tumours

A cytological grading for mast cell tumours (MCTs) would be highly desirable, allowing to select the most appropriate therapeutic intervention prior to surgery. This study evaluates the applicability on fine‐needle aspirations (FNAs) of the novel Kiupel grading system, based on number of mitoses, multinucleated cells, bizarre nuclei and presence of karyomegaly. Fifty consecutive cases with pre‐operative cytological diagnosis were included. In cytological specimens, approximately 1000 cells were evaluated, and the histological grade was assessed on the corresponding resected specimens. On cytology, the above parameters were significantly different between histologically low‐grade and high‐grade tumours (P < 0.001). The cytograding correctly predicted the histological grade in 47 cases (accuracy, 94%; sensitivity, 84.6%; specificity, 97.3%). Two high‐grade MCTs (4%) were not detected on cytology. The cytograding can provide helpful insights to assist clinical decisions in most cases. However, the risk of underestimation in a minority of patients represents a limit to the overall utility of the technique.     

Immunohistochemical detection of p53 tumor-suppressor protein is a poor indicator of prognosis for canine cutaneous mast cell tumors

Eighty-three canine cutaneous mast cell tumors were graded histologically and evaluated immunohistochemically for p53 tumor-suppressor protein expression. An avidin-biotin immunohistochemical protocol incorporated a rabbit polyclonal antibody (CM-1) directed against normal and mutant p53 protein. Positive staining was observed in 44.6% (37/83) of tumors and included 50% (12/24) of grade I (well differentiated) tumors, 46.9% (23/49) of grade II (intermediate differentiation) tumors, and 20% (2/10) of grade III (poorly differentiated) tumors. A statistically significantly higher proportion (P < 0.019) of tumors from the head and neck (83.3%, 10/12), stained positive for p53 than tumors from the thorax, back, abdomen, and axilla (39.4%, 13/33), legs (35.7%, 10/28), or prepuce, scrotal, or inguinal areas (44.4%, 4/9). No statistically significant difference between p53 labeling and histologic grade, breed, or tumor size was present. Survival data were available for 53/83 (63.9%) of dogs. Positive reactivity for p53 was observed in 47% (25/53) of tumors within this group, with 57.9% (11/19) of grade I, 43.3% (13/30) of grade II, and 25% (1/4) of grade III tumors labeled. Mean survival time for the 53 dogs was 12.1 months. The median survival time for dogs with grade III tumors or tumors >5 cm was statistically significantly shorter (P < 0.0001) than for dogs with grades I and II or smaller tumors. Although p53 protein abnormalities may play a role in tumor development or behavior in some canine cutaneous mast cell tumors, immunoreactivity was not associated with lack of tumor differentiation, tumor locations previously shown to demonstrate aggressive biological behavior, breed predisposition, or survival times.     

Immunohistochemical evaluation of p53 expression with different antibodies in malignant canine tumours with or without p53 gene mutation

Six monoclonal antibodies and a polyclonal antibody (CM1) were used to investigate the overexpression of p53 protein by immunohistochemistry (IHC) in six sarcomas and 21 mammary carcinomas from 27 dogs. IHC was compared with p53 gene mutation analysis performed on the same samples. Only the monoclonal PAb240, PAb421 and the CM1 antibodies were able to detect expression of canine p53 protein. CM1 was found to give the highest concordance (8/11) between positive expression of the p53 protein by IHC and the presence of a gene mutation. In the samples that were negative for p53 expression by IHC, but contained a p53 gene mutation according to DNA analysis, the mutation often affected the epitopes that could have been recognized by these antibodies. Only one out of 16 tumours without a p53 gene mutation had a weakly positive IHC result. These findings indicate that in these two types of canine tumours, IHC – particularly with CM1 – can detect many alterations in p53 expression owing to a gene mutation. False‐positive results were very infrequent.     

Immunohistochemical detection of Mdm2 and p53 in feline mammary gland tumors

The objective of this study was to evaluate nuclear reactivity of Mdm2 and p53 proteins by immunohistochemical means in feline mammary gland tumors; 12 adenomas which included 6 adenomatous lesions obtained from the tissue adjacent to adenocarcinomas, and 22 adenocarcinomas. Seven adenomas and 18 adenocarcinomas showed moderate or marked Mdm2 reactivity. Sixteen adenocarcinomas showed moderate to marked p53 reactivity, but 9 adenomas showed none. Discordant Mdm2 overexpression was found in 5 adenomas and 3 adenocarcinomas, although co-overexpression of Mdm2 and p53 was found in 15 adenocarcinomas. These results suggest that nuclear overexpression of Mdm2 is present in the tumors of early stage without p53 overexpression and related to feline mammary gland tumorigenesis. Nuclear overexpression of p53 is more frequent in adenocarcinomas, but not in adenomas.     

Immunohistochemical analysis for Mdm2 and p53 proteins in methylcholanthrene-induced mouse rhabdomyosarcomas

3-methylcholanthrene (MC)-induced mouse 10 embryonal (ERSs) and 24 pleomorphic rhabdomyosarcomas (PRSs) of the dermis were examined immunohistochemically for nuclear reactivity of Mdm2, p53, and proliferating cell nuclear antigen (PCNA). ERSs were microscopically present in the rhabdium layer of the dermis from 10 to 13 weeks post injection (PI), and PRSs developed from 13 weeks PI. Moderate to marked Mdm2 reactivity was observed in each of the 10 ERSs, and 23 of the 24 PRSs. Moderate to marked p53 reactivity was observed in 5 of the 10 ERSs, and 19 of the 24 PRSs. p53 reactivity increased in PRSs compared with ERSs. The level of Mdm2 expression was significantly higher compared with p53 expression. Discordant Mdm2 overexpression was observed in 5 ERSs and 5 PRSs, and discordant p53 overexpression was observed in 1 PRSs, although co-overexpression of Mdm2 and p53 was observed in 5 ERSs and 18 PRSs. PCNA reactivity significantly increased in PRSs compared with ERSs. These results suggest that Mdm2 overexpression is an important pathogenic event in MC-induced mouse rhabdomyosarcomas, and its expression may be induced by p53-independent pathway.     

Immunohistochemical expression of dogTERT in canine testicular tumours in relation to PCNA,ki67 and p53 expression.

The objective of the study was to determine the immunohistochemical expression of canine TERT in canine testicular tumours comparing two different antibodies for TERT, and to correlate them with well established markers specific to dividing cells such as PCNA and ki67, and with expression of the p53 tumour suppressor gene. The study included 36 cases of canine testicular tumours, which were categorized as 12 Sertoli Cell Tumours (SCT), 20 seminomas, 3 interstitial cell tumours and 1 mixed germ cell-sex cord stromal tumour (MT). Two antibodies for hTERT were examined; a highly specific TERT antibody, RCK-hTERT, was evaluated for the first time. Immunodetection of RCK-hTERT was observed in 31% of tumours examined (6/20 Seminomas, 4/12 SCT, 1/3 interstitial cell tumour and 0/1 mixed germ cell-sex cord stromal tumour), while the NCL-hTERT in 67% of them (15/20 Seminomas, 6/12 SCT, 3/3 interstitial cell tumour and 0/1 ΜΤ). PCNA immunoreactivity was detected in all cases. Regarding ki67, 3 SCT, 12 seminomas and all interstitial cell tumours showed clear immunoreaction. p53 immunoreactivity was detected in 6 SCT, 15 seminomas and all interstitial cell tumours. The immunohistochemical expression of both TERT antibodies are discussed and compared in order to clarify their potential usefulness in canine testicular malignancies in relation to the expression of well known cell cycle markers. Our results indicate that TERT and PCNA are useful proliferation markers but not helpful to evaluate prognosis. Instead of that ki67 and p53 could be used for predicting aggressiveness in this group of tumours     

Apoptotic intrinsic pathway proteins predict survival in canine cutaneous mast cell tumours

Mast cell tumours (MCTs) are the most frequent canine round cell neoplasms and show variable biological behaviours with high metastatic and recurrence rates. The disease is treated surgically and wide margins are recommended. Adjuvant chemotherapy and radiotherapy used in this disease cause DNA damage in neoplastic cells, which is aimed to induce apoptotic cell death. Resisting cell death is a hallmark of cancer, which contributes to the development and progression of tumours. The aim of this study was to investigate the expression of the proteins involved in the apoptotic intrinsic pathway and to evaluate their potential use as prognostic markers for canine cutaneous MCTs. Immunohistochemistry for BAX, BCL2, APAF1, Caspase‐9, and Caspase‐3 was performed in 50 canine cases of MCTs. High BAX expression was associated with higher mortality rate and shorter survival. BCL2 and APAF1 expressions offered additional prognostic information to the histopathological grading systems. The present results indicate that variations in the expression of apoptotic proteins are related to malignancy of cutaneous MCTs in dogs.     

TSLC1 tumour-suppressor gene expression in canine mast cell tumours

Tumour suppressor in lung cancer-1 (TSLC1) is a tumour-suppressor gene coding for an adhesion molecule that is expressed by mast cells. Reduced TSLC1 expression is associated with a poor prognosis in several human tumours, and this study sought to investigate if TSLC1 expression could be used to predict outcome in dogs with mast cell tumours (MCTs). Sections of MCTs of different tumour grades from 45 dogs (Group 1) were immunohistochemically assessed for TSLC1 and Ki67 expression. In addition, 35 intermediate-grade MCTs (Group 2) from dogs with known clinical follow-up were immunohistochemically stained for TSLC1 and Ki67. The TSLC1 staining intensity was found to strongly inversely correlate with tumour grade for Group 1 (P = 0.002857). For Group 2 there was a trend towards dogs with lower TSLC1 scores being more likely to die from MCT-related disease (P = 0.058). The intensity of TSLC1 staining inversely correlated with Ki67 expression for both groups.     

Vinblastine and prednisolone chemotherapy for surgically excised grade III canine cutaneous mast cell tumours

The effect of treatment with vinblastine and prednisolone chemotherapy in dogs undergoing only surgical excision of Patnaik grade III cutaneous mast cell tumours is reported. Potential explanatory variables were explored using Kaplan–Meier survival analysis with log‐rank tests. During a median follow‐up period of 429 days, the overall median survival time (MST) was not reached (lower 95% CI = 322 days). The 1‐year survival probability was 0.71 (standard error 0.1), remaining unchanged at 2 years. Secondary disease at presentation was an independent risk factor for survival (P= 0.045). The MST of dogs presenting with secondary disease was 322 days, with a lower 95% confidence interval of 142 days and a 1‐year survival of probability of 0.47 (standard error 0.19). Adverse effects were recorded in 6 of the 108 (5.6%) vinblastine doses given. This chemotherapy regimen is a well‐tolerated adjunct to surgery for grade III mast cell tumours and appears to prolong survival compared with that expected with surgery alone.     

Masitinib mesylate for metastatic and non‐resectable canine cutaneous mast cell tumours

Masitinib mesylate is a tyrosine kinase inhibitor approved for the treatment of gross, non‐metastatic grade II and III canine mast cell tumours (MCTs). This study evaluated the use of masitinib as a frontline and rescue agent for metastatic and non‐metastatic canine MCTs. Identification of toxicities and prognostic factors in these dogs was of secondary interest. Twenty‐six dogs were included in this study. The overall response rate to masitinib was 50%. The median survival time for dogs that responded to masitinib was 630 days versus 137 days for dogs that did not respond (P = 0.0033). Toxicity was recorded in 61.5% of treated dogs, but the majority of adverse events were mild and self‐limiting. Response to masitinib, not tumour grade, stage or location, was the most significant prognostic factor for survival in dogs with MCTs.     

Morphometry of canine cutaneous mast cell tumors

Twenty-four canine cutaneous nodules, diagnosed as mast cell tumors by fine-needle aspiration biopsy and confirmed by histopathologic analysis by staining with hematoxylin and eosin (HE) and toluidine blue, were analyzed by computerized nuclear morphometry on panoptic- and HE-stained cytopathology slides. Two hundred nuclei per lesion were examined. The morphometric parameters investigated were nuclear area, mean diameter, perimeter, regularity factor, and ellipticity factor. Lesions were graded as I (well differentiated), II (intermediate differentiation), or III (poorly differentiated) according to the following morphologic features: invasiveness, cellularity and cellular morphology, mitotic index, and stromal reaction. Nuclear morphometric results were then compared with histopathologic grades. Values of nuclear area, mean diameter, and perimeter increased with increase in histopathologic grade, but statistical analysis revealed significant differences only between grades II and III and between grades I and III when HE was used (P < 0.01) and between grades I and III with panoptic stain (P < 0.05). The ellipticity factor and regularity factor did not reveal significant differences between histopathologic grades. The results indicate that nuclear morphometric analysis, in combination with the rapid and inexpensive cytopathology technique, can help in mast cell tumor grading, thus contributing to the establishment of a more precise prognosis and treatment.     

Increased expression of tissue inhibitor of metalloproteinase‐1 correlates with improved outcome in canine cutaneous mast cell tumours

Canine mast cell tumour (MCT) is a biologically heterogeneous disease. The extracellular matrix degradation promoted by matrix metalloproteinases (MMPs) has been studied in an attempt to elucidate the mechanisms involved in the biological behaviour of tumours. The aim of this study was to characterize the expression of MMP‐2 and ‐9 and tissue inhibitors of metalloproteinase (TIMP)‐1 and ‐2 in canine cutaneous MCTs and to evaluate their prognostic values. Immunohistochemical staining for MMP‐2, MMP‐9, TIMP‐2 and TIMP‐1 was performed in 46 canine cases of MCTs. TIMP‐1 expression showed an independent prognostic value for post‐surgical survival and disease‐related mortality. Dogs with MCTs showing less than 22.9% mast cell TIMP‐1 positivity were more prone to die because of the disease and had a shorter post‐surgical survival. This article suggests the involvement of TIMP‐1 in MCT progression, by contributing to a good outcome in patients with MCTs.