Evaluating the relevance of surgical margins. Part one: The problems with current methodology

The goal of cancer surgery is to achieve a “clean” microscopic resection, with no residual tumour remaining in the wound. To achieve that goal, the surgeon typically incorporates a measured buffer of grossly normal tissue about the entire circumference of the tumour. Microscopic analysis of the resection boundaries is then performed to determine if all traces of the tumour have been completely removed. This analysis is thought to provide a surrogate indication as to the likelihood for that tumour to recur after surgery. However, it is recognised that tumour recurrence may not occur even when microscopic evidence of tumour has been identified at the resection margins, and recurrence can also occur when conventional histology has considered the tumour to have been completely removed. The explanations for this dichotomy are numerous and include technical and practical limitations of the processing methodology, and also several surgeon-related and tumour-related reasons. Ultimately, the inability to confidently determine when a tumour has been removed sufficiently to prevent recurrence can impact on the ability to provide owners with confident treatment advice. In this article, the authors describe the challenges with defining the true extent of the tumour margin from the perspective of the surgeon, the pathologist and the tumour. The authors also provide an analysis of why our current efforts to ensure that all traces of the local tumour have been successfully removed may provide an imperfect assessment of the risk of recurrence.     

Feline intestinal sclerosing mast cell tumour: 50 cases (1997–2008)

This case series presents a unique and unreported variant of feline intestinal mast cell tumour recognized at the CSU Veterinary Diagnostic Laboratory. Fifty cases of feline intestinal mast cell tumours described as having a significant stromal component were reviewed. Neoplastic cells formed a trabecular pattern admixed with moderate to abundant dense stromal collagen (sclerosis). Neoplastic cells had poorly discernible intracytoplasmic granules which demonstrated metachromasia with special histochemical stains consistent with mast cell granules. Additionally, a subset of cases stained for mast cell-specific tryptase and c-kit demonstrated positive immunoreactivity. Eosinophilic infiltrates were moderate to marked in almost all cases. Lymph node and hepatic metastases were present in 66% of the cases. Treatment and clinical outcome was available in 25/50 cases. Twenty-three of these patients died or were euthanized within 2 months of initial diagnosis. This is the first case series to characterize a sclerosing variant of intestinal mast cell tumour in the cat which appears to have a high propensity for metastasis and a guarded prognosis.     

PCNA and grade in 13 canine oral squamous cell carcinomas: association with prognosis

This study evaluated the prognosis factors of age, tumour size, anatomic location, histological grade and proliferating cell nuclear antigen (PCNA) expression in 13 dogs with oral squamous cell carcinoma (OSCC) with bone invasion and without signs of lymph node or distant metastasis. All animals were treated with radical excision performed with at least 1 cm margin, based on computed tomography images. In the 2‐year follow‐up, median disease‐free survival was 138 days for dogs with grade 3 tumours and was not reached for those with grade 2 tumours. Grade 3 tumours and PCNA labelling index ≥65% were related with a shorter disease‐free survival time and consequently poor prognosis (p = 0.003 and p = 0.034, respectively). Mean PCNA labelling index was significantly higher in recurrent cases (p = 0.011). Histological grade and PCNA expression may be important prognosis factors in canine OSCC.     

Inter‐ and intra‐rater reliability and agreement in determining subcutaneous tumour margins in dogs

The objective of this prospective study was to evaluate agreement and reliability of calliper‐based measurements of locally invasive subcutaneous malignant tumours in dogs. Four raters measured the longest diameter of 12 subcutaneous tumours (7 soft tissue sarcomas and 5 mast cell tumours) from 11 client‐owned dogs during 3 randomized, blinded measurement trials, both pre‐ and post‐sedation. Inter‐ and intra‐rater reliability was evaluated using intra‐class correlation coefficient (ICC) and agreement was evaluated using Bland‐Altman plots. Inter‐ and intra‐rater reliability was good (ICC range of 0.8694‐0.89520) and excellent (ICC range of 0.9720‐0.9966), respectively. For agreement calculations, an a priori clinically relevant limit of agreement of 10 mm was set. Inter‐ and intra‐rater agreement was unacceptable with inter‐rater limits of agreement ranging from 15.9 to 55.6 mm and intra‐rater limit of agreement ranging from 11.9 to 28.1 mm. Review of the measurement trial photographs revealed that calliper orientation changes were frequent, occurring in 9/12 (75%) and 8/12 (67%) pre‐ and post‐sedation cases. No significant correlation was found between inter‐rater measurement standard deviations and calliper orientation changes or dog body condition score. These findings suggest veterinarians may have poor agreement in determining the gross edge of tumours, which is expected to introduce bias and inconsistency in tumour staging, assessing response to therapy, and surgical margin planning. Due to the potential consequences for veterinary cancer patients, future studies are needed to validate the present findings.     

Prognostic impact of neoadjuvant aglepristone treatment in clinicopathological parameters of progesterone receptor‐positive canine mammary carcinomas

Neoadjuvant treatment of canine mammary carcinomas with the progesterone receptor (PR) antagonist aglepristone has a PR expression‐related inhibiting effect on proliferation index (PI). The aim of this study was to evaluate the effect of the treatment in the disease‐free period (DFP) and overall survival (OS) of canine mammary carcinomas. Fifty female dogs with mammary carcinomas were treated with aglepristone (n = 34) or oil vehicle (n = 16) before surgery (day 15). PR expression and PI were analysed by immunohistochemistry in samples taken at days 1 and 15. Epidemiological and clinicopathological data were assessed. DFP and OS data were retrieved every 4–6 months for at least 24 months after surgery. Aglepristone treatment increased DFP of animals bearing PR+ tumours with size smaller than 3 cm, complex and mixed tumours, with histologic grades I and II, and with PI ≤ 10%. Although further studies are necessary, current evidence points to treatment with aglepristone as useful for the management of canine mammary tumours.     

Cytological lymph node evaluation in dogs with mast cell tumours: association with grade and survival*

The purpose of this retrospective cohort study is to describe the association of cytological assessment of lymph node metastasis with survival and tumour grade in dogs with mast cell tumours. Regional lymph node aspirates of 152 dogs diagnosed with a mast cell tumour were reviewed and classified according to specific cytological criteria for staging. 97 dogs (63.8%) had stage I tumours, and 55 (36.2%) had stage II tumours. Stage II dogs had a significantly shorter survival time than dogs with stage I disease (0.8 and 6.2 years, respectively; P < 0.0001). Dogs with grade III mast cell tumours were more likely to have stage II disease (P = 0.004). These results suggest that cytological evaluation of lymph nodes in dogs with mast cell tumours provides useful and valuable clinical information, and the results correlate with tumour grade and outcome thus providing a practical and non‐invasive method for staging.     

Doxorubicin chemotherapy for presumptive cardiac hemangiosarcoma in dogs†

Sixty‐four dogs were treated with single‐agent doxorubicin (DOX) for presumptive cardiac hemangiosarcoma (cHSA). The objective response rate (CR + PR) was 41%, and the biologic response rate (CR + PR + SD), or clinical benefit, was 68%. The median progression‐free survival (PFS) for treated dogs was 66 days. The median survival time (MST) for this group was 116 days and was significantly improved compared to a MST of 12 days for untreated control dogs (P = 0.0001). Biologic response was significantly associated with improved PFS (P < 0.0001) and OS (P < 0.0001). Univariate analysis identified larger tumour size as a variable negatively associated with PFS. The high rate of clinical benefit and improved MST suggest that DOX has activity in canine cHSA.     

Evaluation of minichromosome maintenance protein 7 as a prognostic marker in canine cutaneous mast cell tumours

Minichromosome maintenance proteins (MCMs) are sensitive markers of cellular proliferation and have been shown to be significant predictors of survival in several human malignancies. MCM7 was evaluated as a prognostic marker in canine cutaneous mast cell tumours (MCTs). MCM7 immunohistochemistry was performed and an index of MCM7-positive cells calculated in dogs with known outcome. The Receiver Operating Characteristics method was used to individuate the best cut-off value of MCM7 score as predictor of survival. Survival analysis and prognostic variables were analysed with statistical methods. Ninety-five dogs were included with 31 dying of MCTs. A value of 0.18 was used as cut-off value of MCM7 score as a binary variable. The median survival time for MCM7 score ≤0.18 was not reached at 3668 days, whereas for MCM7 score >0.18 was 187 days (log-rank test; P < 0.0001). In the multivariable analysis, MCM7 was significantly associated with survival after controlling for age, surgical margins and histological grade (hazard ratio 9.2; P = 0.001).     

Outcome following treatment of soft tissue and visceral extraskeletal osteosarcoma in 33 dogs: 2008–2013

Extraskeletal osteosarcoma (EOS) is a rare, highly malignant mesenchymal neoplasm arising from viscera or soft tissues characterised by the formation of osteoid in the absence of bone involvement. Owing to the rarity of these neoplasms very little information exists on treatment outcomes. The purpose of this study was to describe the outcome following surgical treatment of non‐mammary and non‐thyroidal soft tissue and visceral EOS in dogs. Thirty‐three dogs were identified; the most common primary tumour site was the spleen. Dogs that had wide or radical tumour excision had longer survival times compared with dogs that had only marginal tumour excision performed [median survival time of 90 days (range: 0–458 days) versus median survival time of 13 days (range: 0–20 days)]. The use of surgery should be considered in the management of dogs with non‐mammary and non‐thyroidal soft tissue and visceral EOS.     

The use of novel lymphatic endothelial cell‐specific immunohistochemical markers to differentiate cutaneous angiosarcomas in dogs

Lymphangiosarcomas are uncommon vascular neoplasms that arise from lymphatic endothelial cells (LECs). They efface and replace normal subcutaneous tissue and are characterised by arborising, vascular channels lined by a single layer of pleomorphic endothelial cells and a paucity of erythrocytes. Lymphangiosarcomas are architecturally similar to hemangiosarcomas, a common malignancy of vascular origin arising from blood vascular endothelial cells. Common immunohistochemical markers for vascular endothelium, such as Factor VIII‐related antigen (F8RA) and CD31, have traditionally been used to confirm the diagnosis of tumours of vascular origin. However, these markers fail to differentiate between lymphangiosarcoma and hemangiosarcoma, which often show overlapping morphologic features, disparate clinical behaviour and require different treatment modalities. Here we describe the use of two novel LEC‐specific markers, lymphatic vessel endothelial receptor‐1 (LYVE‐1) and prospero‐related homeobox gene‐1 (PROX‐1), to further differentiate between vascular tumours of lymphatic (lymphangiosarcoma) and blood (hemangiosarcoma) endothelial cell origin in the dog.     

Canine oral mucosal mast cell tumours

Mast cell tumours (MCTs) are the most common cutaneous tumours of dogs, however rarely they can arise from the oral mucosa. This subset of MCT is reported to demonstrate a more aggressive clinical course than those tumours on the haired skin and the authors hypothesised that dogs with oral, mucosal MCT would have a high incidence of local lymph node metastasis at presentation and that this would be a negative prognostic factor. An additional hypothesis was that mitotic index (MI) would be prognostic. This retrospective study examines 33 dogs with MCTs arising from the oral mucosa. The results suggest that oral mucosal MCTs in the dog have a high incidence of lymph node metastasis at diagnosis (55%) which results in a poor prognosis. MI and nodal metastasis is highly prognostic. Loco‐regional progression is common in these patients and dogs with adequate local control of their tumour had an improved outcome. Despite a more aggressive clinical course, treatment can result in protracted survivals, even when metastasis is present.     

Evaluation of optimal water fluoridation on the incidence and skeletal distribution of naturally arising osteosarcoma in pet dogs

Experimental toxicological studies in laboratory animals and epidemiological human studies have reported a possible association between water fluoridation and osteosarcoma (OSA). To further explore this possibility, a case‐control study of individual dogs evaluated by the UC Davis Veterinary Medical Teaching Hospital was conducted using ecologic data on water fluoridation based on the owner's residence. The case group included 161 dogs with OSA diagnosed between 2008–2012. Two cancer control groups included dogs diagnosed with lymphoma (LSA) or hemangiosarcoma (HSA) during the same period (n = 134 and n = 145, respectively). Dogs with OSA were not significantly more likely to live in an area with optimized fluoride in the water than dogs with LSA or HSA. Additional analyses within OSA patients also revealed no significant differences in age, or skeletal distribution of OSA cases relative to fluoride status. Taken together, these analyses do not support the hypothesis that optimal fluoridation of drinking water contributes to naturally occurring OSA in dogs.     

Enrofloxacin enhances the effects of chemotherapy in canine osteosarcoma cells with mutant and wild‐type p53

Adjuvant chemotherapy improves survival time in dogs receiving adequate local control for appendicular osteosarcoma, but most dogs ultimately succumb to metastatic disease. The fluoroquinolone antibiotic enrofloxacin has been shown to inhibit survival and proliferation of canine osteosarcoma cells in vitro. Others have reported that fluoroquinolones may modulate cellular responses to DNA damaging agents and that these effects may be differentially mediated by p53 activity. We therefore determined p53 status and activity in three canine osteosarcoma cell lines and examined the effects of enrofloxacin when used alone or in combination with doxorubicin or carboplatin chemotherapy. Moresco and Abrams canine osteosarcoma cell lines contained mutations in p53, while no mutations were identified in the D17 cells or in a normal canine osteoblast cell line. The addition of enrofloxacin to either doxorubicin or carboplatin resulted in further reductions in osteosarcoma cell viability; this effect was apparent regardless of p53 mutational status or downstream activity.     

Angiofibroma of the nasal cavity in 13 dogs

This case series describes a rare entity, nasal angiofibroma, in 13 dogs that were presented to the University of Wisconsin, School of Veterinary Medicine from 1988 to 2000. All dogs in this case series presented with clinical signs and radiographic changes that were strongly suggestive of a locally invasive neoplasm. However, histopathology completed on transnostral core biopsy samples revealed benign appearing vascular proliferation with secondary lymphosuppurative inflammation was established despite cytologic criteria of malignancy present in five dogs. On the basis of the outcomes in this case series, nasal angiofibroma should be considered a differential for dogs presenting with clinical signs consistent with a malignant nasal tumour.     

A prospective evaluation of the impact of second‐opinion histopathology on diagnostic testing,cost and treatment in dogs and cats with cancer

Second‐opinion histopathology is a common practice in human medicine to avoid unnecessary procedures, costs and to optimize therapy. Histopathology review has been recommended in veterinary oncology as well. In this prospective evaluation of 52 tumours over a 1‐year period, there was diagnostic agreement between first and second opinions in 52% of cases. Twenty‐nine percent of cases had partial diagnostic disagreement, most often a change in grade, tumour subtype or margin status. Nineteen percent had complete diagnostic disagreement, including a change in cell of origin or a change from benign to malignant. Minor disagreements, which would not affect treatment or prognosis, were present in 21% of cases. Major disagreements, which would affect either treatment or prognosis, were present in 37% of cases. Costs of ideal staging and treatment recommendations were considerably different between first and second opinions.     

Identification of occult micrometastases and isolated tumour cells within regional lymph nodes of previously diagnosed non‐metastatic (stage 0) canine carcinomas

Metastatic dissemination of carcinomas to lymph nodes impacts prognosis and treatment recommendations in human and veterinary medicine. Routine histopathologic evaluation of regional lymph nodes involves haematoxylin and eosin (H&E) staining to identify intra‐nodal neoplastic cells; however, identification of small volume metastases (micrometastases and individual tumour cells) may be missed without the aid of immunohistochemistry or additional step‐sections. The aim of this study was to identify occult carcinoma metastases in previously diagnosed non‐metastatic lymph nodes using step‐sections and pancytokeratin (panCK) immunohistochemistry. Samples from 20 regional lymph nodes diagnosed as non‐metastatic were serially sectioned and evaluated with panCK. Of these, 25% (n = 5) contained micrometastases (n = 1) or isolated tumour cells (n = 4). This study demonstrates the increased efficacy of serial step‐sections combined with panCK immunohistochemistry to identify small volume metastases in regional lymph nodes. The prognostic significance of micrometastases and isolated tumour cells in regional lymph nodes warrants further investigation in veterinary medicine.     

Bilateral removal of the mandibular and medial retropharyngeal lymph nodes through a single ventral midline incision for staging of head and neck cancers in dogs: a description of surgical technique

Currently, there is no standard protocol for removal of regional lymph nodes for the staging of head and neck cancers in dogs. Palpation and fine needle aspiration of mandibular lymph nodes are most commonly performed for staging of head and neck cancers. Although cytology is commonly performed for staging of head and neck, cancers histopathology is required for definitive lymph node staging. When regional lymph node biopsy is performed, mandibular lymph nodes are most commonly sampled due to their accessibility. The medial retropharyngeal lymph nodes may be the most relevant draining lymph node of the head and neck, but they are not routinely sampled due to their anatomic location medial to the salivary glands. The technique described here will allow for a standardized surgical approach for the efficient removal of both mandibular and medial retropharyngeal lymph nodes for staging of head and neck tumours via a single ventral midline approach.     

Phase I/II clinical trial of 2-difluoromethyl-ornithine (DFMO) and a novel polyamine transport inhibitor (MQT 1426) for feline oral squamous cell carcinoma

Polyamines are essential for cell proliferation. Their production is dysregulated in many cancers and polyamine depletion leads to tumour regression in mouse models of squamous cell carcinoma (SCC). The purpose of this study was to determine the maximally tolerated dose of the polyamine transport inhibitor, MQT 1426, when combined with the ornithine decarboxylase (ODC) inhibitor, DFMO, and to determine whether this therapy results in reduction in tumour polyamine levels. Thirteen cats with oral SCC received both drugs orally and serial tumour biopsies were obtained for polyamine measurement. Cats were monitored for response to therapy and toxicity. A maximum tolerated dose (MTD) of MQT 1426 when combined with DFMO was determined. Dose-limiting toxicity was vestibular in nature, but was fully reversible. Spermidine and total polyamine levels decreased significantly in tissues, two cats experienced objective tumour regression and six cats had stable disease. These results suggest that further study of polyamine depletion therapies is warranted.     

Phase II study of oral docetaxel and cyclosporine in canine epithelial cancer

The goal of the current study was to determine the efficacy of oral docetaxel in combination with cyclosporine in the treatment of canine epithelial cancer. Requirements for eligibility were histological confirmation of epithelial neoplasia, measurable disease, no chemotherapy treatment within 2 weeks, and a life expectancy of ≥3 months. Fifty‐one dogs were enrolled. All dogs received 1.625 mg kg^?1 of docetaxel with 5 mg kg^?1 of cyclosporine (DT/CSA) by gavage. Ten dogs had progressive disease at 2 weeks, one dog died, and one dog was withdrawn from the study. Thirty‐nine dogs were given a second dose of DT/CSA, three each receiving a third or fourth dose. Eight dogs had a dose reduction (1.5 mg kg^?1) and six dogs had treatment delays primarily for gastrointestinal toxicity. The overall response rate was 16.7% (8/48 had a partial response there were no complete responses). The highest response rate was seen in dogs with oral squamous cell carcinoma (50%; 6/12).     

Ultrasound and computed tomography of the iliosacral lymphatic centre in dogs with anal sac gland carcinoma

In this prospective study, we hypothesized that computed tomography (CT) would identify more normal and abnormal iliosacral lymph nodes (LNs) than abdominal ultrasound in dogs with anal sac gland carcinoma (ASGC). Twelve client‐owned dogs with ASGC but without distant metastasis were enrolled. Abdominal ultrasound and contrast‐enhanced CT scans of the abdomen were obtained. Iliosacral LNs were counted and assessed for location, laterality and size. Significantly (P < 0.00001) more iliosacral LNs were identified with CT (61) than ultrasound (30), including significantly (P = 0.00012) more medial iliac LNs with CT (33) than ultrasound (19). There was no difference in number of internal iliac LNs identified with CT versus ultrasound. Significantly (P = 0.000061) more sacral LNs were identified with CT (15) than ultrasound (0). Ultrasound identified slightly more (7) abnormal iliosacral LNs than CT (5). Contrast CT was able to identify more normal but not more abnormal LNs than ultrasound.