Melanoma

Question 1

Newman et al. 2012. C-kit expression in canine mucosal melanomas

14 benign dermal and 61 malignant mucosal melanocytic tumors examined for c-kit

Melan-A expression?

C-kit expression?

What was associated with longer survival?

c-kit may be altered in canine melanoma and may have potential as prognostic indicator

Answer 1

100% of dermal melanocytes and in 97% in tumors from oral or conjunctival mucosa - confirming melanocytic origin

Stong and diffuse in cytoplasm in 100% dermal melanocytes and in basilar mucosal melanocytes over submucosal neoplasms (44%), junctional (neoplastic) melanocytes (28%) and less commonly neoplastic melanocytes of subepithelial tumors (10%)

Kit expression in resected melanoma

Question 3

Phillips et al. 2012. Evaluation of tyrosinase kinase expression in canine and equine melanocytic tumors

39 canine and 8 equine tumor samples and 10 canine and 6 equine normal tissue samples

Tyrosinase expression in melanocytic tumors compared to normal tissues?

Tyrosinase expression in histologic variants of melanocytic tumors

Correlation between MHC I and tyrosinase expression or tissue histologic classification

Answer 3

High expression

No significant difference

No correlation

Question 4

Cuitino et al. 2012. Lack of prognostic significance of angiogenesis in canine melanocytic tumors

36 cutaneous melanomas (benign MT), 40 cutaneous melanomas (malignant MTs) and 43 oral melanomas

Mean MVD?

Mean EA?

MVD and EA related to survival?

Tumor vascularization higher in melanomas than melanocytomas but no prognostic significance

Answer 4

Highest in oral melanomas

High in cutanoeus melanoma and oral melanoma

Not related to survival for cutaneous and oral MT

Question 5

Dank et al. 2014. Use of adjuvant carboplatin for treatment of dogs with oral malignant melanoma following surgical excision

Median PFS

First PFS event?

Median OS?

Tumor as cause of death?

Answer 5

259 days (8.6 mo)

Local recurrence 41% and metastasis 41%

440 days (14.7 mo)

60%

Question 6

Han et al. 2013. Alteration in E-cadherin/B-catenin expression in canine melanotic tumors

42 primary canine skin and oral melanomas

B-catenin encoded by ctnnb1 gene - altered expression has been implicated in tumor progression

Expression of ctnnb1in oral melanoma compared to normal melanocytes

Expression of E-cad/B-catenin?

Intranuclear B-catenin?

Disruption of E-cad/B-catenin complexes and increased B-catenin may induce tumor progression and malignancy

Answer 6

Higher

Loss of membrance complex and cytoplasmic accumulation in 84%

Detected in all tissues with reduced membrane B-catenin expression

Question 7

Chon et al. 2013. Activation of the canonical Wnt/B-catenin signaling pathway is rare in canine malignat melanoma tissue and cell lines

Canonical Wnt/B-catenin pathway activation is a ____ event in canine oral malignant melanoma tissue and canine malignant cell lines

Answer 7

Rare

Question 8

Wilson-Robles HM. 2015. Identification and evaluation of putative tumor-initiating cells in canine malignant melanoma cell lines

TIC were identified in 2 cell lines - CML 1 and CML6M

CD34- cells expressed stem cell genes, such as Oct4, Nanog, Ptch1- more efficient at making spheres than CD34+ population

Question 9

Noguchi et al. 2013. MicroRNAs as tumor suppressors in canine and human melanoma cells and as a prognostic factor in canine melanomas

Which microRNA was significantly associated with shorter survival time?

Which 2 microRNA were downregulated in canine and human MM cell lines?

Ectopic expression of ____ has significant inhibitory effect on cell growth by targeting ____

miR-203 is new prognostic factor and miR-205 functions as tumor suppressor by targeting erbb3

Answer 9

miR-203

miR-203 and miR-205

miR-205, erbb3

Question 10

Campagne et al. 2013. Canine melanoma diagnosis: RACK1 as a potential biological marker

RACK1 in classification of 19 cutaneous and 5 mucosal melanocytic neoplasms

MITF as marker of normal and transformed melanocytic cells in dog tissues

All control and tumoral samples stained positive for MITF

RACK1 was not detected in healthy skin melanocytes, melanocytic lesion were positive for RACK1 (100%)

All melanomas stained homogeneously for RACK1

All melanocytomas stained heterogeneously for RACK1

Question 11

Fowles et al. 2015. Comparative analysis of MAPK and PI3K/AKT pathway activation and inhibition in human and canine melanoma

Constitutive pathway activation and similar sensitiviy to AZD6244 (MAPK) and rapamycin (PI3K/AKT) was observed in human and canine cells

Question 12

Choisunirachon et al. 2015. Effects of low-dose cyclophosphamide with piroxicam on tumor neovascularization in a canine oral malignant melanoma-xenografted mouse model

After tx with CyLD, px, or combination, growth of tumor was significantly ___ compared to control group at 30 days of treatment

Proliferation index was significantly ___ by all tretament

Which tx reduced MVD and VEGF levels

Which tx reduced proportion of normal vessels and caused an imbalance between VEGF and thrombospondin-1?

Answer 12

Suppressed

reduced

Cyclophosphamide only

Cyclophosphamide only

Question 13

Ottnod et al. 2013. A retrospective analysis of the efficacy of Oncept vaccine for the adjunct treatment of canine oral malignant melanoma

OMM that had achived loco-regional cancer control

30 dogs with stage II and III disease

Answer 13

Dogs that received the vaccine did not achieve greated PFS, DFI or MST than dogs thay did not receive the vaccine

Question 14

Greene et al. 2013. Expression of leptin and iNOS in oral melanoma in dogs

Melanomas in humans is promoted by inflammatory environment that is contributed by leptin and inducible nitric oxide synthase (iNOS)

20 dogs

Leptin was detected in ___ of tumor cells from 11 dogs

Leptin expression was variable

OM with ___ percentage of iNOS positive cells displayed highest MI

Answer 14

>75%

Lowest

Question 15

Ito et al. 2013. The proteosome inhibitor bortezomib inhibits growth if canine malignant melanoma cells in vitro and in vivo

Bortezomib suppresses the constitutively activated ___ pathway

Answer 15

NF-KB

Question 16

Ahn Jo et al. 2013. Anti-tumor effect of adipose tissue derived-mesunchymal stem cells (AT-MSCs) expressing interferon-B and treatment with cisplatin in xenograft mouse model for canine melanoma

AT-MSCs cell therapy vehicles for anti-tumor molecules

IFN-B transduced canine AT-MSC (cAT-MSC-IFN-B) inhibited growth of LMeC canine melanoma cells

cAT-MSC-IFN-B and low-dose cisplatin reduced tumor volume compared with other treatment groups

Question 17

Malho et al. 2013. Investigation of prognostic indicators for human uveal melanoma as biomarkers of canine uveal melanoma metastasis

Which genes demonstrated increased expression in metastasizing uveal melanoma?

Answer 17

HTR2B, FXR1, LTA4H, and CDH1

Question 18

Seo et al. 2014. Antitumor effects of celocoxib in COX-2 expressing and non-expressing canine melanoma cell lines

Celecoxib selective COX-2 inhbitor inhibits cell growth of various human cancers such as MM

Celecoxib has antitumor effects in canine melanoma LMeC and CMeC-1 cells by inducing ___ cell cycle arrest and apoptosis

Answer 18

G1-S cycle

Question 19

Borrego et al. 2016. Neurokinin-1 receptor expression and antagonism by NK-1R antagonist maropitant in canine melanoma cell lines and primary tumor tissues

NK-1R mRNA and protein expression were observed in majority of tumor tissues

NK-1R immunoreactivity in 11 of 15 tumors

Unable to find cell line to recapitulate effect

Maropitant inhibited proliferation and enahanced apoptosis in cell lines, in absence of NK-1R expression may represent off target effects

Question 20

Riccardo et el. 2014. CSPG4-specific immunity and survival prolongation in dogs with oral malignant melanoma immunized with human CSPG4 DNA

Dogs with stage II-III surgically resected CSPG4-positive oral MM subjected to IM plasmid administration

hCSPG4 electrovaccination resulted in longer overall and disease-free survival times in 14 vaccinated dogs as compared to nonvaccinated controls

All dogs developed Abs

7 vaccinated dogs were tested for T-cell response and 2 had IFNg response

Question 21

Breit et al. 2014. Biologic activity of the novel orally bioavailable selective inhibitor of nuclear transport (SINE) KPT-335 against canine melanoma cell lines

Exportin 1 (XPO1) is chaperone protein - export proteins out of nucleus

Upregulated in human cancers and linked to chemoresistance

Characterize biologic activity of SINE, KPT-335, against canine melanoma cell lines

Effect of KP-335 on cell lines?

Effect on XPO1?

Answer 21

Inhibited proliferation, blocked colony formation, induced apoptosis

Downregulated while increasing mRNA, upregulated p53 and p21

Question 22

Boston et al. 2014. Efficacy of systemic adjuvant therapies administered to dogs after excision of oral malignant melanoma: 151 cases

151 OMM treated by excision with or without adjuvant therapies

MST?

Multivariable analysis, poor prognostic indicators?

Survival benefit from adjuvant therapy including vaccination against melanoma or chemotherapy?

Difference in survival time for dogs that received adjuvant therapy?

Answer 22

346 days (1 year)

Tumor size, patient age, intralesional excision (vs. marginal, wide, radical excision)

None

No difference in survival - 335 days for that did and 352 for those that did not

Question 23

Chon et al. 2015. 6-Bromoindirubin-3’oxime (BIO) decreases proliferation and migration of canine melanoma cell lines

BIO is a serine/threonine kinase inhibitor with specificty for GSK-3B inhibition

BIO treatment of melanoma cell lines enahanced B-catenin mediated transcriptional activity, suggesting GSK-3B inhibition, reduced cell proliferation and migration

Question 24

Guth et al. 2014. Comparison of cancer stem cell antigen expression by tumor cell lines and by tumor biopsies from dogs with melanoma and OSA

What CSC antigens were expresses in cell line?

Markers expressed in tumor biopsy samples?

CSC subpopulations are similar in melanoma and OSA

Answer 24

CD44 and CD90

CD133, CD34, CD44, CD24, Oct3/4

Question 25

Noguchi et al. 2016. Analysis of micR-203 in CREB/MITF/RAB27a pathway: comparison between canine and human melanoma cells

miR-203 is downregulated and acts an anti-oncomir in melanoma cells

miR-203 directly targets CREB1 and regulated its downstream targets, MITF and RAB27a

miR-203 suppressed growth of humand and canine melanoma cells and inhibited melanosome transport through suppression of signaling pathway

Question 26

Cancedda et al. 2016. Efficacy and side effects of radiation therapy in comparison with radiation therapy and temozolomide in the treatment of measurable canine malignant melanoma

Adjuvant oral tem (60 mg/m2 for 5 days every 28 days)

15 dogs treated with RT only (group 1) and 12 dogs received tem (group 2)

Overall response rate of group 1 and group 2?

Median time to progression in group 1 and 2?

Survival time?

Post-radiation tem has good safety profile and may improve TTP in MM

Answer 26

87% and 81% - no difference

110 days (3.7 mo) and 205 (6.8 mo) days significantly longer in group 2 than group 1

No difference

Question 27

Tuohy et al. 2014. Outcome following curative-intent surgery for oral melanoma in dogs: 70 cases (1998-2011)

Significant association with PFI and ST?

Administration of adjuvant treatment was associated with?

Presence of metastasis?

Answer 27

Ajuvant therapy, metastatic disease at time of diagnosis, higher tumor stage (III or IV), increased tumor size (> 3cm), sexually intact female dogs

130% increased hazard of diease progression

281% increased hazard of death

Question 28

Paoloni et al. 2015. Defining the pharmacodynamic profile and therapeutic index of NHS-IL12 immunocytokine in dogs with malignant melanoma

IL-12 proinflammatory cytokine that mediates TH1 response and cytotoxic T-cell activation

NHS-1L 12 administered SQ to dogs with melanoma

AEs included?

Serum IL-10 and CD8+ populatipons?

PR were observed in 2 dogs treated with 0.8 mg/m2 and 1.6 mg/m2

Answer 28

Thrombocytopenia, liver enzymopathies, fever, and vasculitis

Increased after tx

Question 29

Przezdziecki et al. 2015. Accuracy of routine cytology and immunocytochemistry in preoperative diagnosis of oral amelanotic melanoms in dogs

Histopathologic differentiation between amelanotic melanoma, sarcoma, poorly differentiated carcinoma is difficult

38 cases

Undifferentiated oral tumors stained with anti-cytokeratin, anti-vimentin, anti-Melan A antibodies

Specificity and sensitivity of combined routine cytology and ICC?

Diagnosis of 38 amelanotic melanoma, sarcoma, carcinom made with?

Sensitivty and specificity of routine cytology for amelanotic melanoma?

Answer 29

100%

cytology and ICC

moderate - 67% and 86%

Question 30

Kawabe et al. 2015. Outcomes of dogs undergoing radiotherapy for treatment of oral malignant melanoma: 111 cases (2006-2012)

Adjunctive treatments included surgery (18), chemotherapy (39), both (27)

MST for dogs with stage I, II, III, IV melanoma

Dogs with stage I disease had prolonged survival

Answer 30

758 days (25 mo), 278 days (9 mo), 163 days (5.4 mo), 80 days (2.7 mo)

Question 31

Tollett et al. 2016. Palliative radiation therapy for solid tumors in dogs: 103 cases (2007-2011)

Tumors included carcinoma, sarcoma, melanoma, primary bone tumor, MCT, ameloblastoma

Overall tumor and clinical response rate?

MST?

Which dogs had longer survival time?

Tumor location and PFS or MST?

Answer 31

75% and 77%

134 days (4.5 mo)

Dogs that had clinical response or SD had significantly longer survival than PD

No affect

Question 32

Sarbu et al. 2017. Safety of adminitering the canine melanoma DNA vaccine to cats with malignant melanoma - a retropective study

Clinical AE rate?

Most common cause of death?

Safe to administer to cats with minimal risk of AEs

Answer 32

11.4% - pain on vaccine administration, muscle fasiculation, transient inappetance, depression, nausea, mild increase in pigmentation

Melanoma

Question 33

Tregiarri et al. 2016. A retrospective review of outcome and survival following surgery and adjuvant xenogenic DNA vaccination in 32 dogs with oral malignant melanoma

Outcome of 32 dogs treated with surgery and DNA vaccine

MST

Median PFS

What factors influenced survival?

Answer 33

355 days (1 yr)

160 days (5.3 mo)

Stage, margins, delay in vaccine did not influence survival or PFS

Question 34

Iussich et al. 2017. PDGFRs expression in dogs affected by malignant oral melanoma: correlation with prognosis

PDGFR co-expression in 48 cases of CMM?

Positivity for PDGFR-a and B receptor?

PDGFR and Ki-67 co-expression?

Answer 34

37.5%

54% and 47.9%

Associated with worse prognosis

Question 35

Piras et al. 2016. Prolongation of survival of dogs with oral malignant melanoma treated by en bloc surgical resection and adjuvant CSPG4-antigen electrovaccinatin

Reported post-surgery 1-year survival rate for oral canine malignant melanoma (cMM) is around 30%; novel treatments are needed as the role of adjuvant chemotherapy is unclear. This prospective study regards adjuvant electrovaccination with human chondroitin sulfate proteoglycan-4 (hCSPG4)-encoded plasmid in 23 dogs with resected II/III-staged CSPG4-positive oral cMM compared with 19 dogs with resected only II/III-staged CSPG4-positive oral cMM.

Vaccination resulted in 6-, 12-, 18- and 24-month survival rate of 95.6, 73.9, 47.8 and 30.4%, respectively [median survival time (MST) 684 days, range 78-1694, 8 of 23 dogs alive] and 6-, 12-, 18- and 24-month disease-free interval (DFI) rate of 82.6, 47.8, 26.1 and 17.4%, respectively (DFI 477 days, range 50-1694).

Non-vaccinated dogs showed 6-, 12-, 18- and 24-month survival rate of 63.2, 26.3, 15.8 and 5.3%, respectively (MST 200 days, range 75-1507, 1 of 19 dogs alive) and 6-, 12-, 18- and 24-month DFI rate of 52.6, 26.3, 10.5 and 5.3%, respectively (DFI 180 days, range 38-1250). Overall survival and DFI of vaccinated dogs was longer in those <20 kg. In vaccinated and non-vaccinated dogs local recurrence rate was 34.8 and 42%, respectively while lung metastatic rate was 39 and 79%, respectively.

Question 36

Skinner et al. 2016. Patterns of LN metastasis identified following bilateral mandibular and medial retropharyngeal lymphadenectomy in 31 dogs with malignacies of the head

How may had contralateral dissemination?

Ipisilateral metastasis?

Answer 36

62%

92%

Question 37

Maekawa et al. 2016. Immunohistochemical analysis of PD-L1 expression in canine malignant cancers and PD-1 expression on lymphocytes in canine oral melanoma

PD-1 was highly expressed on ____ obtained from oral melanoma

Answer 37

Tumor infiltrating lymphocytes - lymphocytes are exhausted

Question 38

Finotello et al. 2016. Immunohistochemical expression of MDR-Pgp 170 in canine cutaneous and oral melanomas: pattern of expression and association with tumor location and phenotype

25 cases of CMM

Expression of Pgp or oral tumors and cutaneous tumors?

Answer 38

Oral tumors more likely to have membranous Pgp expression (100%) than cutaneous tumors (67%)

Cytoplasmic and nuclear Pgp expression could also be identified

Question 39

Verganti et al. 2017. Use of Oncept melanoma vaccine in 69 canine oral malignant melanomas in UK

13 patients with macroscopic disease treated with vaccine alone or in combination therapy, 8 showed clinical response

3 patients with Stage IV survived 171 (~6mo), 178 (~6mo), 288 days (~10 mo)

Question 40

Sarowitz et al. 2019. Outcome and prognostic factors following curative intent surgery for oral tumors in dogs: 234 cases

Median cause specific survival was shortest for?

Local recurrene highest for?

Distant metastasis highest for?

Curative intent surgery resulted in complete surgical margins in?

Answer 40

melanoma (206 days) and OSA (209d)

FSA (54%)

Melanoma (30%)

85%

Question 41

Vinavak et al. 2017. Malignant anal sac melanoma in dogs: eleven cases (2000-2015)

Most common clinical sign?

Treatment included surgery (8), chemo (1), palliative treatment with pain meds and stool softener (2)

Melanoma vaccine following surgery in 3 dogs and chemo in 1 dog

PFS and OS?

Answer 41

Bloody anal sac discharge and perianal licking

Mean 92 dogs and median 107 days

Question 42

Yoshitake et al. 2017. Molecular investigation of the direct anti-tumor effects of NSAID in a panel of canine cancer cell lines

A strong correlation between cellular expression of COX and sensitivity to NSAID treatment

NSAID inhibited cell growth at higher concentration than those required for functional COX inhibition

Microarray data - 5 genes upregulate (SLC16A6, PER2, HTR2B, BRAF), and 4 downregulated (LOC, H2AF, LOC, ANKRD43) by NSAID exposure to melanoma cell lines

In vitro anti-tumor effects of NSAIDs

Question 43

Grimes et al. 2017. Agreement between cytology and histopathology for regional lymph node metastasis in dogs with melanocytic neoplasm

Agreement between cytology and histopathology was poor

Consensus between routine cytology and histopathology for staging of LNs is poor and does not correlate with survival

Question 44

Noguchi et al. 2017. Bosutinib, an SRC inhibitor, induces caspace-independent cell death associated with permeabilization of lysosomal membranes in melanoma cells

Canine and human melanoma cells were treated with Bosu

Bosu induced caspace-independent cell death, and blocked autophagy flux, which resulted from lysosomal dysfunction

Lysosomal dysfunction was due to lysosomal membrane permeabilization (LMP) - release lysosome hydrolases including cathespin B

Question 45

Lee et al. 2017. IQGAP1 is an oncogenic target in canine melanoma

IQGAP1 is expressed and localizes to a similar extent in both human and canine melanoma by qPCR, WB, immunoflourescence

Deletion of IQGAP1 reduced MAPK pathway activation

Question 46

Starkey et al. 2017. Metastasis-associated microRNA expression in canine

Question 47

Chamel et al. 2016. Non-ocular melanomas in cats: a retrospective study of 30 cases

11 had cutaneous non-auricular melanoma, 6 had tumor located on pinna and 13 in oral cavity

Cats with auricular melanoma were ___ than cats with tumors in other location?

Location and presence of clinical signs were _____ significance, but achromic phenotype was associated with ____ prognosis

Survival with surgery?

Answer 47

Younger

Not prognostic, poor

Survived longer than cats that received medical treatment or no treatment

Question 48

Zuleger et al. 2017. Pilot study of safety and feasibility of DNA microseeding for treatment of spontaneous canine melanoma

hyTYR plasmid DNA administered to the skin via microseeding in dogs with spontaneous melanoma

hyTYR expressing cells were observed in biopsies oh hyTYR DNA microseeding sites

Increased humoral anti-huTYR Abs were seen in 2 of 5 evaluable dogs following microseeding compared to baseline

DNA microseeding is well tolerated in companion dogs with melanoma

Question 49

Maekawa et al. 2017. A canine chimeric monoclonal antibody targeting PD-L1 and its clincial efficacy in canine oral malignant melanoma or undifferentiated sarcoma

PD-1 is expressed on T cells and PD-L1 is expressed on various human cancers

Objective tumor response in 7 dogs with OMM? undifferentiated sarcoma in 2 dogs?

Answer 49

14.3%, 50%

Question 50

Abou et al. Immunohistochemical expression of MCAM/CD146 in canine melanoma

Involved in transendothelial migration and signal transduction

33% were negative, 15.7% were weakly positive, 13.7% were moderately positive and 37% strongly positive

Expressed in cutaneous and oral but immunoreactivity higher in oral than cutaneous

Negative in ocular melanomas

Question 51

Jama et al. 2018. Altered nuclear expression of the deubiquitylase BAP1 cannot be used as a prognostic marker for canine melanoma

BRCA-1 associated protein (BAP1) is a deubiuquitylating enyme that belongs in ubiquitin c-terminal hydrolase subfamily

Nuclear BAP1 detected in canine oral melanoma cell lines

100% of tumor cells were positive for nBAP1 expression - cannot be used as prognostic marker for uveal and mucosal melanoma

Question 52

Porcellato et al. 2018. FoxP3 and IDO in canine melanocytic tumors

Increased risk of death was associated with?

Prognostic value for FoxP3 and IDO - 14.7 Foxp3+ cells/hpf, 6.1 IDO+cells/hpf, 12.5% FoxP3+ cells

Answer 52

Higher FoxP3+ cells, higher CD3+ Foxp3+ and higher IDO+ cells

Question 53

Silvestri et al. 2018. Tumor thickness and modified clark level in canine cutanoeus melanocytic tumors

Question 54

Rushton et al. 2017. Mutation analysis and gene expression profiling of ocular melanomas in cats

Gene expression analysis revealed upregulation of KIT and LTA4H and downregulation of GNAQ, GNA11, BRAF, and RASSF1

Question 55

Shin et al. 2018. Anticancer effects of high-dose ascrobate on canine melanoma cell lines

High dose ascorbate induced apoptosis via activation of Bax

Question 56

Zamarian et al. 2019. MicroRNA Expression in Formalin-Fixed, Paraffin-Embedded Samples of Canine Cutaneous and Oral Melanoma by RT-qPCR

Investigate whether miRNA expression could vary in melanoma samples derived from formalin-fixed, paraffin-embedded (FFPE) tissues. The study included 4 groups:

(1) 9 samples of oral canine malignant melanoma, (2) 10 samples of cutaneous malignant melanoma, (3) 5 samples of healthy oral mucosa, and (4) 7 samples of healthy skin.

Cutaneous canine malignant melanoma showed a decrease of the expression level of miR-145 and miR-365 and an increase of miR-146a and miR-425-5p compared to control samples.

MiR-145 was also downregulated in oral canine malignant melanoma.

The miRNAs with decreased expression may regulate genes involved in RAS, Rap1, and transforming growth factor β (TGF-β) signaling pathways, as well as upregulated genes associated with phosphatidylinositol signaling system, adherens junction, and RAS signaling pathways.

In conclusion, miR-145, miR-365, miR-146a, and miR-425-5p were differentially expressed in canine malignant melanoma and healthy FFPE samples, suggesting that they may play a role in canine malignant melanoma pathogenesis.

Question 57

Schmid et al. 2019. Canine oral primary melanoma cells exhibit shift to mesenchymal phenotype and phagocytic behaviour

Canine oral malignant melanoma (COMM) is a potentially lethal cancer disease.

We established primary cell lines from mostly amelanotic primary COMM and metastases and assessed lesions and derived cells for Melan A, PNL2 and CD146 expression. Then, migration and invasion of CD146-enriched vs -depleted COMM cells were analysed. Epithelial-to-mesenchymal transition (EMT) was addressed by Vimentin-staining and MMP2/MMP9 zymography. Phagocytic behaviour was analysed by histopathological examination and phagocytosis assay.

While Melan A- and PNL2-staining yielded inconsistent data, 100% of COMM sections and primary cells showed CD146 expression, suggesting that this protein may serve as a prognostic marker.

An overall correlation between CD146-expression and migration/invasion was not observed.

All primary cell lines consistently expressed Vimentin and secreted biologically active MMP2, indicating that they had undergone EMT. Importantly, COMM sections exhibited cell-in-cell structures, and all primary cell lines exhibited phagocytic activity, supporting the concept that cell cannibalism may have a role in COMM progression.

Question 58

Yoshikawa et al. 2019. Synthetic microRNA-205 exhibited tumour suppression in spontaneous canine malignant melanoma by intratumoral injection

Efficacy of intratumoral administration of synthetic miR-205 for spontaneous CMMs and to evaluate its potential as systemic therapy.

Ten dogs with various stages of MM were treated with miR-205BP/S3 injected into tumours. Adverse effects (AEs) were assessed in accordance with the Veterinary Cooperative Oncology Group-Common Terminology Criteria for Adverse Events (VCOG-CTCAE) v1.1 guidelines.

Five cases attained complete remission (CR), three attained stable disease (SD), and two cases displayed characteristics of progressive disease (PD).

In all cases, no changes were observed in the blood parameters upon miRNA administration, and miR-205BP/S3 administration did not yield any side effects. The present results suggest that intratumoral administration of miR-205BP/S3 is a potentially applicable treatment for canine melanoma.

Question 59

Vargas et al. 2019. Galectin-3 Expression Correlates with Post-surgical Survival in Canine Oral Melanomas

Galectin (Gal)-3 is a prognostic marker for human neoplasms such as thyroid, gastric, colorectal and prostate cancers.

The protein is related to processes that favour cancer progression, such as angiogenesis, proliferation and apoptosis. The aim of the present study was to characterize the immunohistochemical expression of Gal-3 in canine oral melanomas and to compare it with post-surgical survival, the expression of apoptosis-related proteins and other known prognostic tools.

Twenty-seven samples of canine oral melanomas were evaluated for Gal-3, B-cell lymphoma (BCL) 2, caspase (CASP) 3 and Ki67 expression, mitotic index and degree of nuclear atypia.

Gal-3 cytoplasmic positivity was correlated positively, while nuclear positivity was correlated negatively, with survival.

The intensity of BCL2 labelling was also correlated positively with Gal-3 cytoplasmic positivity. Higher nuclear atypia was observed in dogs with melanoma that died due to the tumour, as well as in dogs that survived for <1 year after surgery. We have confirmed the importance of nuclear atypia for MMs and suggest that Gal-3 is a valuable prognostic indicator for this neoplasm. More in-depth studies are needed to unveil Gal-3 functions in canine MMs using larger sample sizes.

Question 60

Hutchinson et al. 2019. Characterization of myeloid-derived suppressor cells and cytokines GM-CSF, IL-10 and MCP-1 in dogs with malignant melanoma receiving a GD3-based immunotherapy

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells of myeloid origin with immunosuppressive capabilities, which are increased in many human cancers and contribute to tumor immune evasion. They are a possible target to improve immunotherapy outcomes.

Current information regarding MDSCs in canines is minimal, limiting their use as translational model for the study of MDSCs.

The objective of this study was to characterize major MDSCs subsets (monocytic and polymorphonuclear) and the cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 10 (IL-10) and monocyte chemoattractant protein-1 (MCP-1) in canines with malignant melanoma and to evaluate changes in MDSCs and the cytokines over time in response to a GD3-based active immunotherapy.

Whole blood and serum collected from 30 healthy controls and 33 patients enrolled in the University of Florida melanoma vaccine trial were analyzed by flow cytometry with canine specific CD11b, MHCII and anti-human CD14 antibodies to assess ostensibly polymorphonuclear-MDSC (CD11b+ MHCII- CD14-) and monocytic-MDSC (CD11b+ MHCII- CD14+) subsets. IL-10, MCP-1 and both MDSCs subsets were significantly elevated in melanoma dogs versus controls.

Both MDSCs subsets decreased significantly following GD3-based immunotherapy administration but no significant changes in cytokines were seen over time.

To our knowledge, this is the first report documenting increased monocytic-MDSCs in canine melanoma. This is consistent with human malignant melanoma data, supporting dogs as a valuable model for therapeutic intervention studies.

Question 61

Silvestri et al. 2019. Tumor Thickness and Modified Clark Level in Canine Cutaneous Melanocytic Tumors

Breslow thickness and Clark level are prognostic factors for human cutaneous melanomas. Breslow thickness is measured with an ocular micrometer from the top of the granular layer of the epidermis to the deepest invasive cell across the broad base of the tumor, while Clark level is based on the anatomical level of invasion through the layers of the dermis. Because of the anatomical differences between humans and dogs, we evaluated the tumor thickness and a modified Clark level in 77 canine primary cutaneous melanocytic tumors. Tumor thickness (using both a traditional and a more convenient system) and modified Clark level were measured and associated with histological diagnosis and clinical outcome. Tumor thickness was a prognostic factor, being greater in animals with shorter overall survival and disease-free time. Cutoffs of 0.95 cm and 0.75 cm defined a higher hazard for an unfavorable outcome and to develop recurrence/metastasis, respectively. Because of an excellent agreement between the 2 methods, it was concluded that tumor thickness could be measured with a ruler when an ocular micrometer is not available. Modified Clark level was not found to be relevant for prognosis. However, we suggest that both tumor thickness and a modified Clark level can be valid additional parameters when histological diagnosis is uncertain. Further studies, including a wider sample population, would be worthwhile to confirm the prognostic significance of these 2 parameters.

Question 62

Rushton et al. 2019. Circulating cell-free DNA does not harbour a diagnostic benefit in cats with feline diffuse iris melanomas

Objectives: Feline diffuse iris melanoma (FDIM) is the most common malignant primary intraocular tumour in cats, with reported metastases rates between 19% and 63%. Currently, the only available diagnostic tool for a tentative diagnosis is histopathological examination of the enucleated eye. Therefore, the veterinary ophthalmologist is often faced with the dilemma of whether to enucleate an oftentimes visual eye or to continue monitoring, with the risk of metastases developing. In the past, cell-free DNA (cfDNA) gained more attention in human medicine, especially in the field of oncology. Prior studies have shown the use of cfDNA as diagnostic or prognostic markers in canine and human cancer patients. Therefore, the aim of this study was to investigate cfDNA concentration and integrity in cats with FDIMs compared with cats with benign iris naevi and without ocular abnormalities.

Methods: cfDNA from plasma of cats with iris melanoma (n = 34), iris naevus (n = 30) and without ocular abnormalities (n = 32) were extracted. Primer and probes for feline amyloid beta precursor protein ( APP) and beta actin ( ACTB) were designed for amplicons of various lengths and quantitative PCRs of extracted cfDNA were performed to measure cfDNA concentration and integrity of the plasma samples. Differences of cfDNA concentrations and integrity levels between the three groups (iris melanoma, iris naevi and controls) were analysed using the Mann-Whitney U-test.

Results: cfDNA concentration and integrity analysis revealed no significant differences between the cats with iris melanoma, iris naevus or the control group ( P >0.01). Cats with metastases showed similar cfDNA concentration and integrity to cats without metastases.

Conclusions and relevance: cfDNA concentration and integrity seem to be insufficient as a diagnostic or prognostic marker in cats with FDIMs.

Question 63

Grimes et al. 2019. Histologic evaluation of mandibular and medial retropharyngeal lymph nodes during staging of oral malignant melanoma and squamous cell carcinoma in dogs

Objective: To assess histologic evaluation of mandibular lymph nodes (MLNs) and medial retropharyngeal lymph nodes (MRLNs) for metastatic disease during tumor staging for dogs with oral malignant melanoma (OMM) and oral squamous cell carcinoma (OSCC).

Design: Retrospective multi-institutional study.

Animals: 27 dogs with OMM and 21 dogs with OSCC.

Procedures: Medical record databases of 8 institutions were searched to identify dogs with OMM or OSCC that underwent unilateral or bilateral extirpation of the MLNs and MRLNs during the same procedure between January 2004 and April 2016. Information extracted from the records included signalment, primary mass location and size, diagnostic imaging results, histologic results for the primary tumor and all lymph nodes evaluated, and whether distant metastasis developed.

Results: Prevalence of lymph node metastasis did not differ significantly between dogs with OMM (10/27 [37%]) and dogs with OSCC (6/21 [29%]). Distant metastasis was identified in 11 (41%) dogs with OMM and was suspected in 1 dog with OSCC. The MRLN was affected in 13 of 16 dogs with lymph node metastasis, and 3 of those dogs had metastasis to the MRLN without concurrent metastasis to an MLN. Metastasis was identified in lymph nodes contralateral to the primary tumor in 4 of 17 dogs that underwent contralateral lymph node removal.

Conclusions and clinical relevance: Results indicated histologic evaluation of only 1 MLN was insufficient to definitively rule out lymph node metastasis in dogs with OMM or OSCC; therefore, bilateral lymphadenectomy of the MLN and MRLN lymphocentra is recommended for such dogs.

Question 64

Souza et al. 2019. entral mandibulectomy for removal of oral tumours in the dog: Surgical technique and results in 19 cases

The purpose of this retrospective study is to describe in detail a novel ventral approach for mandibulectomy and the results in 19 dogs. The medical records of 19 dogs that received a partial or total unilateral mandibulectomy with the new ventral approach were reviewed. Information obtained included signalment, tumour type, extent of mandibulectomy, removal of regional lymph nodes, intrasurgical complications, immediate postoperative complications, histopathological diagnosis and study of margins. Intrasurgical complication occurred in one dog (haemorrhage) and required a blood transfusion. Postoperative morbidity was minor and included transient ventral cervical swelling and self-limiting sublingual swelling (two dogs). All 19 animals were discharged between 24 and 48 hours of the procedure, and appetite was considered normal at discharge. Some perceived advantages of this procedure include easy identification of all the important anatomic structures in the area, including the inferior alveolar artery and temporo-mandibular joint, and the fact that osteotomy of the zygomatic arch is not necessary (in case of caudal mandibulectomy). In addition, dissection of both mandibular and retropharyngeal lymph nodes is easily achieved by caudal extension of the same skin incision.

Question 65

Kbayashi et al. Tissue factor procoagulant activity in the tumor cell lines and plasma of dogs with various malignant tumors

Hypercoagulability is a common paraneoplastic complication in dogs with various malignant tumors. Importantly, tissue factor procoagulant activity (TF-PCA) induced by TF-bearing microparticles (TF-MPs) is associated with hypercoagulability in human patients with cancer. However, TF-PCA in tumor cells and the association between circulating TF-MPs and hypercoagulability in dogs with malignant tumors remain poorly understood. Therefore, the present study was conducted to evaluate the TF-PCA in various types of canine tumor cell lines and plasma in dogs with malignant tumors. Mammary gland tumor, hemangiosarcoma, and malignant melanoma cell lines, but not lymphoma cell lines, expressed TF on their surfaces and showed cellular surface and MP-associated TF-PCA. The plasma TF-PCA was elevated in some dogs that naturally developed such tumors. No significant difference was observed in plasma TF-PCA between the disseminated intravascular coagulation (DIC) group (median: 43.40; range: 3.47-85.19; n=5) and non-DIC group (median: 7.73; range: 1.70-16.13; n=12). However, plasma TF-PCA was remarkably elevated in three of five dogs with DIC. To the best of our knowledge, this is the first study to evaluate plasma TF-PCA in dogs with malignant tumors. Further studies must be conducted to determine the cellular origin of TF-MPs and the efficacy of plasma TF-PCA as a biomarker of DIC in dogs with malignant tumors.