Treatment of nasal tumours in dogs: a review.

Nasal tumours are common neoplasms in dogs and often represent a diagnostic and therapeutic challenge due to their confined location within the nasal cavities. The main goal of this review is to extract the most relevant information from a wide and often confusing evidence-based medicine on the treatment of canine nasal tumours and conclude with current recommendations. This report highlights the different therapeutic modalities available and describes their technical aspects, interests and limitations. Megavoltage radiotherapy, as the most recent treatment and standard of care, is particularly examined, especially the different types of radiotherapy units, the main protocols used and their advantages and limits. Newer and non-conventional treatments are also discussed.

Toxicity of cytarabine constant rate infusion in dogs with high-grade non-Hodgkin lymphoma with bone marrow or central nervous system involvement.

OBJECTIVE: Cytarabine, a cell-cycle phase-specific antimetabolite, has been reported to improve outcomes in dogs with bone marrow (BM) or central nervous system (CNS) lymphoma involvement receiving combination chemotherapy. The objective of this study was to evaluate the incidence and severity of toxicity of cytarabine constant rate infusion (CRI) in dogs with high-grade non-Hodgkin lymphoma. METHODS: Medical records of canine lymphoma patients with confirmed or suspected BM (group 1) or CNS (group 2) involvement, treated with a modified cyclophosphamide, epirubicin, vincristine and prednisolone protocol, including a single dose of cytarabine given as CRI, were reviewed and adverse events graded. RESULTS: Twenty-six dogs were included. Gastrointestinal toxicity occurred in 17 dogs (65.3%), with 5 (19.2%) experiencing grade III or IV toxicity. Neutropenia occurred in nine dogs (34.6%), but was grade I or II in most cases. Three dogs (11.5%) had thrombocytopenia: one grade III and two grade IV. Four dogs (15.3%) experienced increases in alanine amino transferase: one each grade I and II and two grade III. Five dogs (19.2%) required hospitalisation to manage toxicity after completing cytarabine CRI, and haematological toxicity resulted in treatment delays in five dogs (median delay of 4 days, range: 3-7 days). CONCLUSION: Our findings suggest that gastrointestinal toxicity should be expected in lymphoma patients undergoing cytarabine CRI.

Haematologic toxicity in dogs with mast cell tumours treated with vinblastine/prednisolone chemotherapy with/without radiotherapy.

OBJECTIVES: To determine whether dogs with surgically excised mast cell tumours receiving a vinblastine/prednisolone chemotherapy protocol in combination with radiation therapy are at greater risk of myelosuppression than patients receiving the chemotherapy protocol alone. MATERIALS AND METHODS: Retrospective study of clinical records of dogs with mast cell tumours that, subsequent to surgical excision, had received combination vinblasine/prednisolone chemotherapy. Dogs were assigned to two groups: those treated with adjunctive radiotherapy and vinblastine/prednisolone (RT group) and those treated with surgery followed by vinblastine/prednisolone alone (control group). Haematology results were compared between groups. RESULTS: Forty-three cases and 43 controls of similar breed, age and bodyweight were included. Concurrent radiation and vinblastine chemotherapy did not appear to increase the risk of neutropenia, which was observed in 18.6 and 23.2% of cases in the RT and control groups, respectively. CLINICAL SIGNIFICANCE: Radiation and vinblastine chemotherapy can be safely combined in dogs with mast cell tumours without increasing the risk of clinically important myelosuppression.

Genome-wide analysis of canine oral malignant melanoma metastasis-associated gene expression.

Oral malignant melanoma (OMM) is the most common canine melanocytic neoplasm. Overlap between the somatic mutation profiles of canine OMM and human mucosal melanomas suggest a shared UV-independent molecular aetiology. In common with human mucosal melanomas, most canine OMM metastasise. There is no reliable means of predicting canine OMM metastasis, and systemic therapies for metastatic disease are largely palliative. Herein, we employed exon microarrays for comparative expression profiling of FFPE biopsies of 18 primary canine OMM that metastasised and 10 primary OMM that did not metastasise. Genes displaying metastasis-associated expression may be targets for anti-metastasis treatments, and biomarkers of OMM metastasis. Reduced expression of CXCL12 in the metastasising OMMs implies that the CXCR4/CXCL12 axis may be involved in OMM metastasis. Increased expression of APOBEC3A in the metastasising OMMs may indicate APOBEC3A-induced double-strand DNA breaks and pro-metastatic hypermutation. DNA double strand breakage triggers the DNA damage response network and two Fanconi anaemia DNA repair pathway members showed elevated expression in the metastasising OMMs. Cross-validation was employed to test a Linear Discriminant Analysis classifier based upon the RT-qPCR-measured expression levels of CXCL12, APOBEC3A and RPL29. Classification accuracies of 94% (metastasising OMMs) and 86% (non-metastasising OMMs) were estimated.

Acute radiotherapy toxicity in 57 dogs with gross and microscopic mast cell tumours.

Mast cell tumours (MCTs) are commonly treated with radiation therapy, most often in a microscopic disease setting. Poorer outcomes are expected in patients with gross disease, and irradiation of gross disease may be associated with greater toxicity. The aim of this study was to compare acute radiation adverse events (AE) in dogs with gross and microscopic MCTs receiving radiotherapy. Fifty-seven dogs were included, 28 with gross disease and 29 with microscopic. In order to assess mucosal and skin toxicity, patients were assigned to 2 groups: head (29 patients, 14 patients with gross and 15 microscopic) and other sites (28 patients, 14 each). All were treated with external beam radiotherapy, and toxicity assessed at the end of treatment and 10 to 14 days later (first recheck). All patients developed some acute radiation toxicity by the end of the course. However, there was no difference in the severity of toxicity between gross and microscopic disease in either site group at either time point. The only variable associated with an increased frequency of grade 2 or 3 toxicity at the first recheck was the use of prednisolone prior to radiotherapy (P = .05). No other factors were identified which were associated with increased toxicity. For the head group, the site of highest grade toxicity was mucosa or, if included in the field, nasal planum, which was often more severely affected than the mucosa. No significant late toxicity was identified. Two dogs developed acute haematemesis during the radiotherapy course, but both completed the course without further events.

Evaluation of a multi-agent chemotherapy protocol combining lomustine, procarbazine and prednisolone (LPP) for the treatment of relapsed canine non-Hodgkin high-grade lymphomas.

The standard of care treatment for canine lymphoma is multi-agent chemotherapy containing prednisolone, cyclophosphamide, vincristine and an anthracycline such as doxorubicin (CHOP) or epirubicin (CEOP). Lomustine, vincristine, procarbazine, and prednisone (LOPP) has been evaluated as a rescue, with encouraging results; however, resistance to vincristine is likely in patients relapsing on CHOP/CEOP, and this agent may enhance LOPP toxicity without improving efficacy. The aim of this study was to evaluate responses to a modified-LOPP protocol that does not include vincristine (LPP) and is administered on a 21-day cycle. Medical records of dogs with high-grade multicentric lymphoma from 2012 to 2017 were reviewed. Dogs with relapsed lymphoma that received LPP as a rescue protocol were enrolled. Response, time from initiation to discontinuation (TTD) and toxicity of LPP were assessed. Forty-one dogs were included. Twenty-five dogs (61%) responded to LPP including 12 complete responses (CR) and 13 partial responses (PR). Responders had a significantly longer TTD (P < .001) compared to non-responders with 84 days for CR and 58 days for PR. Neutropenia was documented in 20 dogs (57%): 12 grade I to II, 8 grade III to IV. Thrombocytopenia was infrequent (20%): 5 grade I to II, 2 grade III to IV. Twelve dogs developed gastrointestinal toxicity (30%): 10 grade I to II and 2 grade III. Nineteen dogs had elevated ALT (59%): 9 grade I to II, 10 grade III to IV. Treatment was discontinued due to toxicity in 8 dogs (19%). The LPP protocol shows acceptable efficacy and toxicity-profile and minimizes in-hospital procedures.

A preliminary investigation of the effect of sample collection technique on the cell and RNA content of fine-needle aspirates of five canine tumours.

OBJECTIVES: To evaluate the effect of syringe size, needle size, number of needle passes and operator experience on cell yield from tumour fine-needle aspirates, and the quantity and quality of extractable RNA. MATERIALS AND METHODS: Fine-needle aspirates were collected from canine lymphoma, cutaneous mast cell tumour, anal gland adenocarcinoma, fibrosarcoma and oral malignant melanoma using nine different techniques. RESULTS: There was a significant difference in cell yield between fine-needle aspirate techniques for melanoma, lymphoma and anal gland adenocarcinoma. The application of suction yielded the largest number of cells. Cell numbers in lymphoma and fibrosarcoma aspirates collected by different veterinary surgeons were not significantly different. Use of a smaller gauge needle and suction increased the quantity of RNA isolated from fibrosarcoma and anal gland adenocarcinoma aspirates, but did not influence RNA integrity. CLINICAL SIGNIFICANCE: Suction during fine-needle aspiration increases cell numbers obtained from five common canine tumours. Suction increases the quantity of RNA isolated from anal gland adenocarcinoma and fibrosarcoma aspirates without affecting RNA quality. Junior veterinary surgeons gain comparable cell numbers to senior staff.

Epirubicin in the treatment of canine histiocytic sarcoma: sequential, alternating and rescue chemotherapy.

The aims of this study were to report treatment outcomes for dogs with histiocytic sarcoma (HS) treated with both lomustine and epirubicin, and to report response rates to epirubicin as a rescue therapy in dogs previously treated with lomustine. Medical records of dogs with a diagnosis of HS that were treated with both lomustine and epirubicin were retrospectively evaluated. Of 29 dogs receiving epirubicin alternating with, or subsequent to lomustine treatment, including in a rescue setting, response to epirubicin could be assessed in 20 with an overall response rate (ORR) of 29% and biological response rate (BRR) of 71%. Median time to progression (TTP) in 12 of these 20 dogs in which it was assessable was 69 days (range: 40-125 days). For dogs treated in the rescue setting epirubicin specific ORR was 19% and BRR 63%. Median TTP in the 9 of these 16 dogs in which it was assessable was 62 days (range: 40-125 days). Median survival time for all dogs treated with both epirubicin and lomustine was 185 days (range: 27-500 days). Some dogs with HS respond to epirubicin and dogs treated with combinations of epirubicin and lomustine have modestly improved survival times compared with single agent studies, and similar to dogs with HS treated with alternating lomustine and doxorubicin. Single agent epirubicin is also a valid short term rescue therapy for canine HS.

Temozolomide alone or in combination with doxorubicin as a rescue agent in 37 cases of canine multicentric lymphoma.

Temozolomide (TMZ) is an alkylating agent previously used in conjunction with doxorubicin (DOX) to treat dogs with relapsed lymphoma. However, there are very limited data for this drug when used as single agent. The aim of this retrospective study was to evaluate the efficacy and toxicity of TMZ in dogs with relapsed multicentric lymphoma that failed multi-agent chemotherapy protocols, and compare the outcome to a group of dogs receiving the same drug in combination with DOX. Twenty-six patients were included in the TMZ group and 11 in the TMZ/DOX group. Responses were evaluated via retrospective review of the medical records. The overall median survival time (MST) for both groups was 40 days (range 1-527 days). For the TMZ group, median time to progression (TTP) was 15 days (range 1-202 days) and MST 40 days (range 1-527 days), with an overall response rate (ORR) of 32% and 46% recorded toxicities. For the TMZ/DOX group, median TTP was 19 days (range 2-87 days) and MST 24 days (range 3-91 days), with an ORR of 60% and 63% recorded toxicities. However, a proportion of haematological toxicoses may have gone undetected due to the absence of associated clinical signs. The difference in MST and TTP between the 2 groups was not statistically significant. Similarly, no negative prognostic factors were identified. Although responses were generally short lived, this study suggests that TMZ may achieve similar efficacy to TMZ/DOX whilst being associated with a lower frequency of recorded toxicities.

Use of Oncept melanoma vaccine in 69 canine oral malignant melanomas in the UK.

OBJECTIVES: Oral malignant melanomas carry a poor-to-guarded prognosis because of their local invasiveness and high metastatic propensity. The Oncept melanoma vaccine is licensed to treat dogs with stage II or III locally-controlled oral malignant melanoma and this retrospective study aimed to assess survival of affected dogs treated with the vaccine in the UK. MATERIAL AND METHODS: Medical records of dogs with histopathologically-confirmed oral malignant melanoma that received the vaccine as part of their treatment were evaluated. Survival analyses for potential prognostic factors were performed. RESULTS: Sixty-nine dogs were included; 56 dogs, staged I to III, and with previous locoregional therapy, had a median survival time of 455 days (95% CI: 324 to 586 days). Based on Kaplan-Meier survival analysis with associated log-rank testing, no significant prognostic factors were identified for this population. Of the 13 patients with macroscopic disease treated with vaccine alone or in combination therapy, eight showed clinical response. Three patients with stage IV oral malignant melanoma survived 171, 178 and 288 days from diagnosis. CLINICAL SIGNIFICANCE: Patients treated with the melanoma vaccine in our study had survival times similar to their counterparts receiving the vaccine in the USA. There were observed responses in patients with macroscopic disease and so the vaccine could be considered as palliative treatment in dogs with stage IV disease.

Toxicity of metronomic cyclophosphamide chemotherapy in a UK population of cancer-bearing dogs: a retrospective study.

OBJECTIVES: The objective of this study was to assess the incidence of toxicity in a group of cancer-bearing dogs treated with metronomic chemotherapy. MATERIALS AND METHODS: Retrospective review of dogs treated with metronomic doses of cyclophosphamide: between 5 and 15 mg/m2 /day or every other day for treatment of neoplasia. RESULTS: Of the 65 dogs included, there were signs of, mostly mild, toxicity in 32 (49%). The most common toxicities were sterile haemorrhagic cystitis (n=16) and gastrointestinal disorders (n=12). Median time to development of sterile haemorrhagic cystitis was 110 days (range 7 to 686 days). Four dogs developed suspected bacterial infections during treatment. CLINICAL SIGNIFICANCE: Metronomic cyclophosphamide is generally well-tolerated in dogs but the incidence of sterile haemorrhagic cystitis in this study is higher than previously reported. Regular urinalysis is recommended for all dogs receiving cyclophosphamide chemotherapy, as early detection of haemorrhagic cystitis may prevent development of more serious disease.

A descriptive review of cardiac tumours in dogs and cats.

Cardiac tumours are uncommon in the canine and feline population and often an incidental finding. Common types include haemangiosarcoma (HSA), aortic body tumours (chemodectoma and paraganglioma) and lymphoma. These neoplasms can cause mild to severe, life-threatening clinical signs that are independent of the histological type and may be related to altered cardiovascular function or local haemorrhage/effusion into the pericardial space. Cardiac tumours may require symptomatic treatment aimed at controlling tumour bleeding and potential arrhythmias, and other signs caused by the mass effect. Additional treatment options include surgery, chemotherapy and radiotherapy. For all medical therapies, complete remission is unlikely and medical management, beyond adjunctive chemotherapy in HSA, requires further investigation but combination chemotherapy is recommended for lymphoma. The aim of this report is to summarize and critically appraise the current literature in a descriptive review. However, interpretation is limited by the lack of definitive diagnosis and retrospective nature of most studies.

Post-radiotherapy hypothyroidism in dogs treated for thyroid carcinomas.

Hypothyroidism is a common adverse event after head and neck radiotherapy in human medicine, but uncommonly reported in canine patients. Records of 21 dogs with histologically or cytologically confirmed thyroid carcinoma receiving definitive or hypofractionated radiotherapy were reviewed. Nine cases received 48 Gy in 12 fractions, 10 received 36 Gy in 4 fractions and 2 received 32 Gy in 4 fractions. Seventeen cases had radiotherapy in a post-operative setting. Ten cases developed hypothyroidism (47.6%) after radiotherapy. The development of hypothyroidism was not associated with the radiotherapy protocol used. Median time to diagnosis of hypothyroidism was 6 months (range, 1-13 months). Hypothyroidism is a common side effect following radiotherapy for thyroid carcinomas. Monitoring of thyroid function following radiotherapy is recommended. No specific risk factors have been identified.

Expression of VEGFR and PDGFR-α/-ß in 187 canine nasal carcinomas.

Radiotherapy represents the standard of care for intranasal carcinomas. Responses to tyrosine kinase inhibitors (TKIs) have been reported but data on expression of target receptor tyrosine kinases (rTKs) is limited. This study characterizes the expression of vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR)-α and PDGFR-ß in canine intranasal carcinomas. Histological samples from 187 dogs were retrieved. Immunohistochemistry was performed using commercially available antibodies. Expression of rTKs was classified into weak, moderate or intense and additionally recorded as cytoplasmic, membranous, cytoplasmic-membranous, nuclear or stromal. VEGFR was expressed in 158 dogs with predominantly moderate expression (36.9%) and a cytoplasmic-membranous expression pattern (70.9%). PDGFR-α was detected in 133 with predominantly weak expression (57.9%) and cytoplasmic pattern (87.9%). PDGFR-ß was identified in 74 patients with a predominantly moderate expression (17.6%) and cytoplasmic expression pattern (63.5%). Co-expression of rTKs was common. These results confirm expression of VEGFR, PDGFR-α and PDGFR-ß in canine intranasal carcinomas and support the utility of TKIs.

Clinical outcome, PDGFRß and KIT expression in feline histiocytic disorders: a multicentre study.

Information about histiocytic disease in cats is limited. The aim of this study was to document clinical findings and outcome in feline histiocytic disorders, and characterize the expression of PDGFRß and KIT in order to identify potential treatment targets. Morphologically diagnosed feline histiocytic tumours were reviewed and characterized by immunohistochemistry (IHC). Five cases of feline progressive histiocytosis (FPH), eight histiocytic sarcomas (HS) and two haemophagocytic histiocytic sarcomas (HaeHS) were confirmed. PDGFRß was variably positive in most histiocytic cases, while KIT was negative in all. Clinical presentation, treatment and outcome were also evaluated. Partial responses were recorded in measurable disease with tyrosine kinase inhibitors and lomustine, and radiotherapy achieved long-term control in some cases. Survival times were shortest in HaeHS and disseminated disease. PDGFRß, but not KIT, may represent a therapeutic target in feline histiocytic disorders but more studies are needed to investigate other potential treatment targets.

Quantifying alcohol-related emergency admissions in a UK tertiary referral hospital: a cross-sectional study of chronic alcohol dependency and acute alcohol intoxication.

OBJECTIVES: Alcohol is responsible for a proportion of emergency admissions to hospital, with acute alcohol intoxication and chronic alcohol dependency (CAD) implicated. This study aims to quantify the proportion of hospital admissions through our emergency department (ED) which were thought by the admitting doctor to be (largely or partially) a result of alcohol consumption. SETTING: ED of a UK tertiary referral hospital. PARTICIPANTS: All ED admissions occurring over 14 weeks from 1 September to 8 December 2012. Data obtained for 5497 of 5746 admissions (95.67%). PRIMARY OUTCOME MEASURES: Proportion of emergency admissions related to alcohol as defined by the admitting ED clinician. SECONDARY OUTCOME MEASURES: Proportion of emergency admissions due to alcohol diagnosed with acute alcohol intoxication or CAD according to ICD-10 criteria. RESULTS: 1152 (21.0%, 95% CI 19.9% to 22.0%) of emergency admissions were thought to be due to alcohol. 74.6% of patients admitted due to alcohol had CAD, and significantly greater than the 26.4% with 'Severe' or 'Very Severe' acute alcohol intoxication (p<0.001). Admissions due to alcohol differed to admissions not due to alcohol being on average younger (45 vs 56 years, p<0.001) more often male (73.4% vs 45.1% males, p<0.001) and more likely to have a diagnosis synonymous with alcohol or related to recreational drug use, pancreatitis, deliberate self-harm, head injury, gastritis, suicidal ideation, upper gastrointestinal bleeds or seizures (p<0.001). An increase in admissions due to alcohol on Saturdays reflects a surge in admissions with acute alcohol intoxication above the weekly average (p=0.003). CONCLUSIONS: Alcohol was thought to be implicated in 21% of emergency admissions in this cohort. CAD is responsible for a significantly greater proportion of admissions due to alcohol than acute intoxication. Interventions designed to reduce alcohol-related admissions must incorporate measures to tackle CAD.

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.

Canine pancreatic islet cell tumours secreting insulin-like growth factor type 2: a rare entity.

Insulin-like growth factor type II (IGF-II) is the main cause of non-islet cell tumour hypoglycaemia (NICTH) and insulin is thought to be the only factor causing hypoglycaemia in insulinomas. However, two case reports of pancreatic neuroendocrine tumours (PNETs) producing IGF-II have been previously published: a human and a canine patient. In this study, we investigated clinical, histopathological, immunohistochemical and ultrastructural features, and biological behaviour of canine pancreatic IGF-II-omas, a subgroup of PNETs that has not been previously characterized. Case records of 58 dogs with confirmed PNETs and hypoglycaemia were reviewed: six patients were affected by IGF-II-omas. Surgery was performed in all cases and two dogs had metastases. Four patients remained alive and in remission at 370, 440, 560 and 890 days post-diagnosis; two died of non-tumour-related causes. IGF-II-omas can be differentiated from insulinomas through hypoinsulinaemia, IGF-II positive and insulin negative immunostaining. The prevalence of this neoplasia is low, accounting for just 6% of PNETs.

The diagnostic utility of lymph node cytology samples in dogs and cats.

OBJECTIVES: The aim of this study was to determine common reasons for lymph node fine needle aspirates, cytological diagnoses reported and the frequency and reasons for non-diagnostic samples from dogs and cats. METHODS: Retrospective study of computerised records of fine needle aspirate samples submitted to NationWide Laboratories (UK) between April 2009 and May 2011 to identify lymph node samples. Reason for sampling, sample quality, diagnosis achieved and reason for non-diagnostic samples were assessed. RESULTS: A total of 1473 records were available for review. Of 1274 canine samples, 928 (72 · 8%) were diagnostic and 346 (27 · 2%) were non-diagnostic. Of 199 feline samples, 171 (85 · 9%) samples were diagnostic and 28 (14 · 1%) were non-diagnostic. The most common reasons for sample submission in both species were investigation of lymphadenopathy (alone or in combination with other clinical signs) or tumour staging. In dogs, the most common diagnosis was lymphoma (351, 27 · 5%), and in cats, reactive hyperplasia (63, 31 · 6%). Absence of cells, cell disruption and low yield were the most common causes of non-diagnostic samples. Submission of the history did not affect the probability of reaching a cytological diagnosis. CLINICAL SIGNIFICANCE: Lymph node cytology is a useful diagnostic procedure but educating veterinarians to improve sampling and smearing may increase diagnostic yield.

Prospective study to investigate the use of fine needle aspiration techniques in UK veterinary practice.

OBJECTIVES: To document the fine needle aspiration methods used by UK veterinary practitioners for the assessment of cutaneous masses and relate this to the achievement of a representative sample. METHODS: An internet-based questionnaire was designed and publicised in the UK national veterinary press, at a national surgical meeting, and in letters to veterinary surgeons. RESULTS: One hundred and seventy respondents replied to the questionnaire: 58 · 2% sampled cutaneous masses on the basis of appearance or behaviour; 41 · 3% sampled every cutaneous mass. Practitioners with a greater oncological caseload or who graduated more recently were more likely to recommend fine needle aspiration for every cutaneous mass (P = 0 · 019 and P = 0 · 0002 respectively); 66 · 5% of respondents applied suction during fine needle aspiration; 89% of all respondents used a 2 or 5 mL syringe in combination with a 21 or 23 G needle. There was no statistically significant association between achievement of a representative sample and syringe (P = 0 · 64) or needle size (P = 0 · 63). CLINICAL SIGNIFICANCE: Fine needle aspiration is widely used in UK practice, but may be underutilised in practices with lower oncological caseloads. Survey participants reported a high rate of representative samples obtained using all the commonly used techniques. Further work is required to confirm these observations.