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Supplemental Figures S1-S4 + two supp. tables

Background Domestic rabbits (Oryctolagus cuniculi) can develop a variety of cutaneous neoplasms, including squamous cell carcinoma (SCC). A detailed review of gross and microscopic pathology and response to treatment of spontaneously arising SCCs in domestic rabbits has not been published. Methods A retrospective survey study of the clinical characteristics and response to treatment in 39 cases of spontaneous SCC in pet rabbits was performed in an attempt to better characterize the typical presentation, prognosis, and therapeutic response of SCCs in domestic rabbits. Sixteen of these cases were also selected for papillomavirus testing using a generic polymerase chain reaction. Results SCC was identified in rabbits between 2 and 10 years of age, with a median age of 7 years. The neoplasm has a predilection for ears and feet and the conventional subtype is most commonly diagnosed microscopically. Lighter colored rabbits may be predisposed to developing SCC. The majority of cases examined were found in rabbits housed primarily indoors. Only one SCC tested positive for papillomavirus and was located in the oral cavity. Sequencing of the detected PCR product detected 98.75% similarity to human papillomavirus type 120. The significance of this virus for tumorigenesis is unknown. Conclusions and clinical relevance Aggressive surgical resection provided the most successful therapeutic option and proved curative in 12 of 23 rabbits. Papillomavirus likely does not play a major role in the development of spontaneous SCCs in pet rabbits. More research is needed to investigate the use of adjunctive therapies in treatment of this neoplasm in pet rabbits.

c-Kit mutations have been reported in 15% to 40% of certain human melanoma subtypes, including those histologically similar to canine oral malignant melanomas. Therapeutic response to tyrosine kinase inhibitors has been demonstrated in those human patients. As canine oral malignant melanomas tend to have a poor prognosis despite aggressive surgical removal, evaluation of KIT expression and identification of c-Kit mutations in canine oral melanocytic neoplasms was performed to determine if there is any indication that tyrosine kinase inhibitor drugs might effectively treat any of these cases. This study evaluated 27 canine oral malignant melanomas and 12 canine histologically well-differentiated oral melanocytic neoplasms for activating c-Kit mutations, determined differences in immunohistochemical expression of KIT and c-Kit mutation status, and determined if KIT expression could predict c-Kit mutation status. Among samples that contained intraepithelial nests of neoplastic melanocytes in the KIT-labeled sections, KIT was expressed within cells in these nests in 22/23 (96%) malignant melanomas and 5/7 histologically well-differentiated neoplasms. KIT was expressed in 10% to 30% of neoplastic melanocytes in the lamina propria in 3/24 (13%) malignant melanomas, but 0/9 (0%) histologically well-differentiated neoplasms. Next-generation sequencing identified 85 variants in c-Kit, including 9 nonsynonymous mutations that resulted in amino acid changes predicted to affect protein function. c-Kit mutations with predicted deleterious protein effects were more common in malignant melanomas (8/27 [30%] vs 1/12 [8%]). There was no apparent relationship between detected c-Kit mutations and KIT expression. These results do not support the use of therapies that target c-Kit.

Canine pituitary dwarfism in German Shepherd and related dog breeds has been reported to be associated with a 7-bp deletion mutation in intron 5 of the LHX3 gene. This mutation is transmitted as an autosomal recessive trait that results in dwarf dogs with significantly smaller stature and abnormal haircoat, and potentially early death. Phenotypically, affected adult dogs are proportionally dwarfs. These dwarfs also have a soft, woolly puppy coat that fails to transition into the typical adult hair coat, and marked hair loss occurs in some dogs. We report a similar manifestation of dwarfism in Tibetan Terriers with the same LHX3 mutation. Dwarf Tibetan Terrier puppies were born physically normal but failed to gain weight or to grow at the same rate as their normal littermates. The 7-bp deletion mutation of the LHX3 gene was identified in both alleles of 3 Tibetan Terrier dwarfs from 3 litters, which were biologically related. All parents of these dogs are carriers, confirming transmission of dwarfism in an autosomal recessive manner. Recognition and detection of this mutation will help in guiding future breeding plans to eventually eliminate this trait from Tibetan Terriers.

Bernese mountain dogs (BMDs), have an overall cancer incidence of 50%, half of which is comprised of an otherwise rare tumor, histiocytic sarcoma (HS). While recent studies have identified driver mutations in the MAPK pathway, identification of key predisposing genes has been elusive. Studies have identified several loci to be associated with predisposition to HS in BMDs, including near the MTAP/CDKN2A region, but no causative coding variant has been identified. Here we report the presence of a coding polymorphism in the gene encoding FANCG, near the MTAP/CDKN2A locus. This variant is in a conserved region of the protein and appears to be specific to BMDs. Canine fibroblasts derived from dogs homozygous for this variant are hypersensitive to cisplatin. We show this canine FANCG variant and a previously defined hypomorphic FANCG allele in humans impart similar defects in DNA repair. However, our data also indicate that this variant is neither necessary nor sufficient for the development of HS. Furthermore, BMDs homozygous for this FANCG allele display none of the characteristic phenotypes associated with Fanconi anemia (FA) such as anemia, short stature, infertility, or an earlier age of onset for HS. This is similar to findings in FA deficient mice, which do not develop overt FA without secondary genetic mutations that exacerbate the FA deficit. In sum, our data suggest that dogs with deficits in the FA pathway are, like mice, innately resistant to the development of FA.

Canine cutaneous mast cell tumors (ccMCTs) have a highly variable biological behavior and accurate prognostication is essential for therapeutic intervention. Internal tandem duplications (ITD) of exon 11 are the most commonly detected c-kit mutation in ccMCTs and are associated with poor prognosis and increased cellular proliferation. The prognostic value of detecting mutations in other exons of c-kit has not been systematically examined. In this study, we analyzed the prognostic value of ITD mutations of exon 8 in c-kit of ccMCTs in comparison to ccMCTs with ITD mutations of exon 11 and ccMCTs without mutations of exon 8 or 11. The mutational status, histological grade, KIT expression pattern, Ki67 index, AgNOR (argyrophilic nucleolar organizing region) score, and Ag67 score were determined in 221 ccMCTs, and outcome was available for 101 dogs. ITD mutations of exon 8 were found in 73/221 (33%), of exon 11 in 100/221 (45%), and none of these mutations in 50/221 (22%) of ccMCTs. None of the dogs with mutations of exon 8 died due to suspected ccMCT-related cause, but 23% dogs with ccMCTs with mutations of exon 11 died due to suspected ccMCT-related cause. Prognostic parameters in ccMCTs with exon 11 mutations were commonly associated with a high proliferative activity and poor prognosis, while prognostic markers in ccMCTs with mutations of exon 8 had lower values similar to those observed in ccMCTs without mutations in exons 8 or 11 of c-kit. This study indicates that screening for ITD mutations in exon 8 in ccMCTs may be helpful to identify less aggressive ccMCTs and may be recommended as a supplementary prognostic test.

An 11-day-old little blue penguin ( Eudyptula minor) died unexpectedly. Prior to hatching, the egg experienced trauma and resultant defects were repaired. The chick hatched without complication and was clinically normal prior to death. Necropsy revealed congested lungs. Histologic examination showed moderate nonsuppurative encephalitis with focally extensive neuronal necrosis and intranuclear inclusions in neurons within necrotic foci. Herpesvirus DNA was detected in brain tissue with a generic herpesvirus polymerase chain reaction. Sanger sequencing demonstrated 100% and 98% sequence homology to sphenicid alphaherpesvirus 1 and penguin herpesvirus 2, respectively. In situ hybridization demonstrated large amounts of herpesvirus nucleic acid in intranuclear inclusions and neuronal nuclei. Combined histology, polymerase chain reaction, Sanger sequencing, and in situ hybridization results were most consistent with herpesviral encephalitis, most likely caused by sphenicid alphaherpesvirus 1. To our knowledge, this is the first report of a herpesvirus infection causing encephalitis in a penguin and the first report of herpesvirus in this species.

The mitotic count (MC) is an important histological parameter for prognostication of malignant neoplasms. However, it has inter- and intraobserver discrepancies due to difficulties in selecting the region of interest (MC-ROI) and in identifying or classifying mitotic figures (MFs). Recent progress in the field of artificial intelligence has allowed the development of high-performance algorithms that may improve standardization of the MC. As algorithmic predictions are not flawless, computer-assisted review by pathologists may ensure reliability. In the present study, we compared partial (MC-ROI preselection) and full (additional visualization of MF candidates and display of algorithmic confidence values) computer-assisted MC analysis to the routine (unaided) MC analysis by 23 pathologists for whole-slide images of 50 canine cutaneous mast cell tumors (ccMCTs). Algorithmic predictions aimed to assist pathologists in detecting mitotic hotspot locations, reducing omission of MFs, and improving classification against imposters. The interobserver consistency for the MC significantly increased with computer assistance (interobserver correlation coefficient, ICC = 0.92) compared to the unaided approach (ICC = 0.70). Classification into prognostic stratifications had a higher accuracy with computer assistance. The algorithmically preselected hotspot MC-ROIs had a consistently higher MCs than the manually selected MC-ROIs. Compared to a ground truth (developed with immunohistochemistry for phosphohistone H3), pathologist performance in detecting individual MF was augmented when using computer assistance (F1-score of 0.68 increased to 0.79) with a reduction in false negatives by 38%. The results of this study demonstrate that computer assistance may lead to more reproducible and accurate MCs in ccMCTs.

The mitotic count (MC) is an important histological parameter for prognostication of malignant neoplasms. However, it has inter- and intra-observer discrepancies due to difficulties in selecting the region of interest (MC-ROI) and in identifying/classifying mitotic figures (MFs). Recent progress in the field of artificial intelligence has allowed the development of high-performance algorithms that may improve standardization of the MC. As algorithmic predictions are not flawless, the computer-assisted review by pathologists may ensure reliability. In the present study we have compared partial (MC-ROI preselection) and full (additional visualization of MF candidate proposal and display of algorithmic confidence values) computer-assisted MC analysis to the routine (unaided) MC analysis by 23 pathologists for whole slide images of 50 canine cutaneous mast cell tumors (ccMCTs). Algorithmic predictions aimed to assist pathologists in detecting mitotic hotspot locations, reducing omission of MF and improving classification against imposters. The inter-observer consistency for the MC significantly increased with computer assistance (interobserver correlation coefficient, ICC = 0.92) compared to the unaided approach (ICC = 0.70). Classification into prognostic stratifications had a higher accuracy with computer assistance. The algorithmically preselected MC-ROIs had a consistently higher MCs than the manually selected MC-ROIs. Compared to a ground truth (developed with immunohistochemistry for phosphohistone H3), pathologist performance in detecting individual MF was augmented when using computer assistance (F1-score of 0.68 increased to 0.79) with a reduction in false negatives by 38%. The results of this study prove that computer assistance may lead to a more reproducible and accurate MCs in ccMCTs.

Supplemental Material, sj-pdf-1-vet-10.1177_03009858211009784 for Correlation Between KIT Expression and c-Kit Mutations in 2 Subtypes of Canine Oral Melanocytic Neoplasms by Rebecca C. Smedley, Tuddow Thaiwong, Lorna E. Deeth and Matti Kiupel in Veterinary Pathology

Epizootic epitheliotropic disease virus (salmonid herpesvirus-3; EEDV) is responsible for the death of millions of hatchery-raised lake trout (

PI3K/AKT/mTOR pathway hyperactivation is frequent in T-cell acute lymphoblastic leukemia/lymphoma (T-ALL/LBL). To model inhibition of mTOR, pre–T-cell lymphoblastic leukemia/lymphoma (pre-T LBL) tumor development was monitored in mice with T lymphocyte–specific, constitutively active AKT (Lck-MyrAkt2) that were either crossed to mTOR knockdown (KD) mice or treated with the mTOR inhibitor everolimus. Lck-MyrAkt2;mTOR KD mice lived significantly longer than Lck-MyrAkt2;mTOR wild-type (WT) mice, although both groups ultimately developed thymic pre-T LBL. An increase in survival was also observed when Lck-MyrAkt2;mTOR WT mice were treated for 8 weeks with everolimus. The transcriptional profiles of WT and KD thymic lymphomas were compared, and Ingenuity Pathway Upstream Regulator Analysis of differentially expressed genes in tumors from mTOR WT versus KD mice identified let-7 and miR-21 as potential regulatory genes. mTOR KD mice had higher levels of let-7a and miR-21 than mTOR WT mice, and rapamycin induced their expression in mTOR WT cells. CDK6 was one of the most downregulated targets of both let-7 and miR21 in mTOR KD tumors. CDK6 overexpression and decreased expression of let-7 in mTOR KD cells rescued a G1 arrest phenotype. Combined mTOR (rapamycin) and CDK4/6 (palbociclib) inhibition decreased tumor size and proliferation in tumor flank transplants, increased survival in an intravenous transplant model of disseminated leukemia compared with single agent treatment, and cooperatively decreased cell viability in human T-ALL/LBL cell lines. Thus, mTOR KD mice provide a model to explore drug combinations synergizing with mTOR inhibitors and can be used to identify downstream targets of inhibition.

An 11-day-old little blue penguin (

A 15-y-old Miniature horse mare had a 6-mo history of an ulcerated mass on the right lower eyelid. An incisional biopsy and a subsequent excisional biopsy were submitted to the Michigan State University Veterinary Diagnostic Laboratory for microscopic evaluation. Histologically, the incisional biopsy was composed of sheets of large neoplastic vacuolated polygonal cells. A few regions contained poorly differentiated neoplastic round-to-basaloid cells that rimmed the sheets of highly vacuolated polygonal cells. Both vacuolated and basaloid cells exhibited strong perimembranous and cytoplasmic immunoreactivity for E-cadherin and cytokeratin 5/6, respectively. Vacuolated polygonal cells were histochemically negative for periodic acid–Schiff, mucicarmine, and oil red O, consistent with a diagnosis of poorly differentiated carcinoma. Within the excisional biopsy specimen, there were anastomosing cords and nests of neoplastic squamous epithelial cells that merged with sheets of similar vacuolated polygonal cells. These findings are consistent with a squamous cell carcinoma with clear cell differentiation. In addition, in the adjacent dermis, there was solar elastosis suggestive of ultraviolet (UV) damage. A clear cell variant of squamous cell carcinoma is a rare entity in humans that previously has not been described in animals, to our knowledge, and is often associated with chronic UV exposure.

Primary colorectal follicular lymphomas are rare indolent lymphoid neoplasms in humans that have not been reported in dogs. We describe 3 cases of primary colorectal follicular lymphoma in dogs with histologic and immunohistochemical features similar to their human counterpart. Initial clinical signs in all dogs included tenesmus, hematochezia, and a palpable rectal mass. Two dogs were castrated males and 1 an intact female, between 9 months and 2 years of age, and of varied breeds. All 3 cases of colorectal follicular lymphoma were characterized by proliferation of follicular germinal centers with no polarity or mantle zone and were composed of centrocytes admixed with fewer centroblasts. By immunohistochemistry, lymphoid cells expressed CD20, BCL2, and BCL6 and lacked expression of CD3, CD5, and cyclin D1. Polymerase chain reaction for rearrangements of the immunoglobulin heavy chain confirmed a monoclonal population in all cases. In 2 of the 3 cases, a solitary nodular colorectal mass was excised and appeared curative; however, the third case had multiple colorectal masses and the animal developed multicentric lymphoma. This case series immunohistochemically characterizes and distinguishes colorectal follicular lymphoma from atypical lymphoid hyperplasia.

Recently, histologic subtypes of oral squamous cell carcinoma (SCC) corresponding to the human classification scheme have been proposed for dogs. A papillary squamous cell carcinoma subtype is characterized by dominant exophytic architectural growth with limited invasion, a lower metastatic rate, and better overall survival compared with conventional SCC. Whereas most canine oral conventional SCCs are easily diagnosed by histologic examination, the diagnosis of canine oral papillary squamous cell carcinoma (COPSCC) can be challenging since the exophytic portion lacks histologic features of malignancy and appears similar to oral nonviral papillomas. In contrast, the invasive portion of COPSCC has morphologic similarities to conventional SCC and canine acanthomatous ameloblastoma. The goals of this study were to immunophenotype these 3 entities and to potentially identify discriminating markers. A panel of 17 immunohistochemical markers was investigated in tissue microarrays that included 25 COPSCCs, 10 conventional SCCs, and 10 canine acanthomatous ameloblastomas. Additionally, COPSCCs were screened for papillomavirus as a potential cause using immunohistochemistry and in situ hybridization. COPSCC had immunophenotypical similarities with conventional SCC and acanthomatous ameloblastoma, but the combined differences in immunolabeling for AE1/AE3, 34βE12, p63, and calretinin discriminated between the entities. Papillomavirus was not detected in any COPSCC, making a viral pathogenesis unlikely. A better understanding of the immunophenotype of COPSCC will aid in a more accurate diagnosis and potentially improve therapeutic approaches.

Histiocytic sarcoma (HS) is an aggressive malignant neoplasm of dendritic cell origin that is common in certain breeds of dogs. High prevalence of fatal, disseminated HS has been described in Bernese Mountain Dogs (BMDs). Support for genetic predisposition to develop HS has been presented in several studies, but to date, causative genetic events have not been reported. In addition, no driver mutations have been identified in tumours. Recently, E76K gain-of-function mutation in SHP2 encoded by the PTPN11 gene has been described in human histiocytic malignancies. In our study, we identified the PTPN11E76K in HS of BMDs. Amplification of exon 3 of the PTPN11 gene followed by Sanger sequencing was used to detect the mutation and estimate the prevalence in HS from 30 BMDs, 13 Golden Retrievers and 10 other dog breeds. The overall prevalence of PTPN11E76K in HS of BMDs was 36.67% compared with 8.69% in other breeds. No mutation was identified in normal tissues from 10 BMDs with HS that carried the mutation and 12 control dogs with no neoplastic disease, including 6 BMDs. Increased immunoreactivity for AKT, phosphorylated ERK1/2 and phosphorylated AKT in a small subset of BMDs with PTPN11E76K suggests that a gain-of-function might be mediated by the ERK and AKT pathways. These data suggest PTPN11E76K as an important driver mutation of HS in BMDs. This information may not only aid in unravelling the tumourigenic events associated with HS in BMDs, but also help in identifying more promising therapeutic strategies.

Combined_supplemental_materials-Shavalier_et_al for Disease Progression in Lake Trout (Salvelinus namaycush) Experimentally Infected With Epizootic Epitheliotropic Disease Virus (Salmonid Herpesvirus-3) by Megan Shavalier, Mohamed Faisal, Thomas P. Loch, Scott D. Fitzgerald, Tuddow Thaiwong and Matti Kiupel in Veterinary Pathology

Supplemental Material, Combined_supplemental_materials-Tuxbury_et_al for Herpesvirus Encephalitis in a Little Blue Penguin (Eudyptula minor) by Kathryn A. Tuxbury, Charles J. Innis, Tuddow Thaiwong, Annabel G. Wise, Roger Maes, Michael M. Garner and Matti Kiupel in Veterinary Pathology

Anaplastic large T-cell lymphoma (ALTCL) is a rare subtype of non-Hodgkin T-cell lymphoma that occasionally occurs in the gastrointestinal tract of humans. Enteropathy-associated T-cell lymphoma (EATL) type 1 is the most common type of intestinal lymphoma in dogs, and ALTCL has not previously been reported in the intestinal tract of dogs. Thirteen dogs with intestinal masses diagnosed as intestinal lymphoma with anaplastic morphology were reviewed. Clinical data, including treatment protocols, were available for 11 cases. Immunohistochemistry for CD3, CD20, and CD30 was performed for all cases in addition to PCR for Antigen Receptor Rearrangements (PARR) for assessment of clonality. Eight (62%) of the cases presented with intestinal perforation, and all cases had 1 or more masses arising from the small intestine. Histologically, all cases were characterized by transmural infiltrates of large, CD3-positive and frequently CD30-positive cells. Neoplastic T cells had marked anisocytosis and anisokaryosis, prominent nucleoli, and occasionally indented to reniform nuclei. There was abundant necrosis and inflammation with occasional vascular invasion within neoplastic masses. All cases had a monoclonal T-cell receptor γ gene rearrangement. The median survival time was 5 days, with 1 dog surviving 2 years after the initial diagnosis. ALTCL can occur as an aggressive transmural lymphoma in the gastrointestinal tract of dogs and commonly causes intestinal perforation. ALTCL can be differentiated from EATL type 1 and might have implications for accurate prognostication and selection of therapeutic options in the future.

While the genetic contributions to the predisposition of Bernese mountain dogs (BMDs) to histiocytic sarcoma (HS) remains unclear, some insights into key genetic drivers have been gained. Our group recently reported a mutation in the PTPN11 gene (E76K). We have now identified a second missense mutation in PTPN11 (G503V), and a mutation in KRAS (Q61H) present in HS cell lines. These mutations are associated with malignancies in humans, and known to be gain-of-function mutations that result in activation of the mitogen-activated protein kinase (MAPK) pathway. The goal of the present study was to evaluate the prevalence of these mutations in a large sample of HS cases from BMDs and golden retrievers, and in lymphoma cases, from a cohort of BMDs. Mutations in PTPN11 were present in HS in 41/96 (43%) BMDs, and in 3/13 (23%) golden retrievers. PTPN11 mutations E76K and G503V did not coexist in the same neoplasm. The KRAS mutation was much less frequent, with a prevalence of 3.1% (3/96). We did not identify either PTPN11 nor KRAS mutations in any of the lymphoma samples. These results point out the potential relevance of PTPN11 and KRAS mutations as activators of the oncogenic MAPK pathway for canine HS, particularly in BMDs.

Recurrent outbreaks of sudden death and bloody diarrhea were reported in March 2013 and February 2014 in a breeding colony of Papillon dogs. During the first outbreak, 1 adult dog and 2 eight-month-old puppies died. During the second outbreak, 2 ten-week-old puppies died. One puppy from the first outbreak and 2 puppies from the second outbreak were examined at necropsy. Histologically, all 3 puppies had severe segmental crypt necrosis of the small intestine and marked lymphoid follicle depletion in the spleen and Peyer’s patches. Real-time (RT) polymerase chain reaction (PCR) demonstrated abundant canine parvovirus (CPV-2) DNA (Ct

Supplemental Material, Combined_supplemental_materials-Bertram_et_al_2 for Computerized Calculation of Mitotic Count Distribution in Canine Cutaneous Mast Cell Tumor Sections: Mitotic Count Is Area Dependent by Christof A. Bertram, Marc Aubreville, Corinne Gurtner, Alexander Bartel, Sarah M. Corner, Martina Dettwiler, Olivia Kershaw, Erica L. Noland, Anja Schmidt, Dodd G. Sledge, Rebecca C. Smedley, Tuddow Thaiwong, Matti Kiupel, Andreas Maier and Robert Klopfleisch in Veterinary Pathology

Previous studies have evaluated cellular proliferation indices, KIT expression, and c-kit mutations to predict the clinical behavior of canine mast cell tumors (MCTs). The study purpose was to retrospectively compare mitotic index, argyrophilic nucleolar organizer regions (AgNORs)/nucleus, Ki-67 index, KIT labeling pattern, and internal tandem duplication mutations in c-KIT between stage I and stage II grade II MCTs. Medical records and tumor biopsy samples from dogs with Grade II MCTs with cytological or histopathological regional lymph node evaluation were included. Signalment, tumor location and stage, and presence of a recurrent versus de novo tumor were recorded. Mitotic index, AgNORs/nucleus, Ki-67, KIT staining pattern, and internal tandem duplication mutations in exon 11 of c-KIT were evaluated. Sixty-six tumors (51 stage I; 15 stage II) were included. Only AgNORs/nucleus and recurrent tumors were significantly associated with stage (odds ratio 2.8, 95% confidence interval [CI] 1.0–8.0, P = .049; odds ratio 8.8, 95% CI 1.1–69.5; P = .039). Receiver-operator characteristic analysis showed that the sensitivity and specificity of AgNORs/cell ≥ 1.87 were 93.3% and 27.4%, respectively, (area under the curve: 0.65) for predicting stage. Recurrent tumors and higher AgNORs/nucleus are associated with stage II grade II MCTs; however, an AgNOR cutoff value that reliably predicts lymph node metastasis was not determined.

Veterinary pathology of infectious, particularly viral, and neoplastic diseases has advanced significantly with the advent of newer molecular methodologies that can detect nucleic acid of infectious agents within microscopic lesions, differentiate neoplastic from nonneoplastic cells, or determine the suitability of a targeted therapy by detecting specific mutations in certain cancers. Polymerase chain reaction–based amplification of DNA or RNA and in situ hybridization are currently the most commonly used methods for nucleic acid detection. In contrast, the main methodology used for protein detection within microscopic lesions is immunohistochemistry. Other methods that allow for analysis of nucleic acids within a particular cell type or individual cells, such as laser capture microdissection, are also available in some laboratories. This review gives an overview of the factors that influence the accurate analysis of nucleic acids in formalin-fixed tissues, as well as of different approaches to detect such targets.

Wohlfahrtiimonas chitiniclastica is an emerging human pathogen that has been identified as the cause of septicemia in humans in Europe and South America. Here we report the first case of a unique disease manifestation of Wohlfahrtiimonas chitiniclastica -induced bacterial septicemia secondary to wound myiasis in a deer in Michigan in the United States.

Objective Advanced systemic mastocytosis (SM) is characterized by uncontrolled growth of neoplastic mast cells (MC) and drug resistance. The tyrosine kinase receptor KIT is often mutated and activated and thus contributes to malignant growth of MC. Therefore, KIT-targeting drugs are currently tested for their ability to block growth of malignant MC. Materials and Methods We determined the effects of the multikinase inhibitor INNO-406 (bafetinib) on primary neoplastic MC, the canine mastocytoma cell line C2, the human MC leukemia cell line HMC-1.1 bearing the KIT mutant V560G, and HMC-1.2 cells harboring KIT V560G and KIT D816V. Results INNO-406 was found to inhibit proliferation in HMC-1.1 cells (IC 50 : 30−40 nM), but not in HMC-1.2 cells or primary neoplastic cells in patients with KIT D816V-positive SM. In canines, growth-inhibitory effects of INNO-406 were seen in C2 cells (IC 50 : 50−100 nM) exhibiting a KIT exon 11 internal tandem-duplication and in primary neoplastic MC harboring wild-type exon 11, whereas no effects were seen in MC exhibiting a polymorphism at amino acid 581 in exon 11. INNO-406 was found to block KIT phosphorylation and expression in HMC-1.1 cells and C2 cells, but not in HMC-1.2 cells, whereas Lyn-phosphorylation was blocked by INNO-406 in all types of MC. Conclusions In neoplastic MC, the major target of INNO-406 appears to be KIT. Drug responses may depend on the presence and type of KIT mutation. In human MC, the KIT D816V mutant introduces resistance, and in canine mastocytomas, an exon 11 polymorphism may be indicative of resistance against INNO-406.

Aggressive mast cell (MC) tumors are hematopoietic neoplasms characterized by uncontrolled growth of MC and resistance to conventional drugs. In most cases, the tyrosine kinase (TK) receptor KIT is involved in malignant cell growth. Therefore, several KIT TK-targeting drugs are currently being tested for their ability to block growth of neoplastic MC. We examined the effects of four TK inhibitors (imatinib, midostaurin, nilotinib, and dasatinib) on C2 canine mastocytoma cells, as well as primary neoplastic canine MC. As assessed by (3)H-thymidine incorporation experiments, all TK inhibitors produced dose-dependent inhibition of proliferation in C2 cells with the following IC(50) values: imatinib: 269 +/- 180 nM, midostaurin: 157 +/- 35 nM, nilotinib: 55 +/- 24 nM, dasatinib: 12 +/- 3 nM. Growth-inhibitory effects of TK inhibitors were also observed in primary neoplastic mast cells, although IC(50) values for each drug varied from patient to patient, with midostaurin being the most potent agent in all samples tested. In consecutive experiments, we were able to show that TK inhibitors cooperate with each other in producing growth inhibition in C2 cells with synergistic effects observed with most drug combinations. In flow cytometry and TUNEL assay experiments, growth-inhibitory effects of TK inhibitors were found to be associated with cell-cycle arrest and apoptosis. Together, these data show that several TK-targeting drugs induce apoptosis and inhibit proliferation in canine mastocytoma cells in vitro, and that synergistic drug interactions can be obtained. Clinical trials are now warranted to explore whether these TK inhibitors also counteract growth of neoplastic cells in vivo in patients with aggressive MC tumors.

The AKT/mTOR pathway is frequently hyperactivated in T-cell acute lymphoblastic leukemia (T-ALL). To model inhibition of this pathway in lymphoma, mice with T-lymphocyte-specific, constitutively active AKT (Lck-MyrAkt2) were crossed to mice with genetically reduced mTOR expression (knock-down, KD). Mice with genetic reduction of mTOR had increased survival by 10 weeks relative to wild type mTOR mice, though both developed thymic pre-T-cell lymphoblastic leukemia/lymphoma (pre-T LBL). Similarly, when mTOR wild type Lck-MyrAkt2 mice were treated for 8 weeks with the rapamycin analog, everolimus, an inhibitor of the mTOR TORC1 complex, survival was also increased. Gene expression profiling of thymic lymphomas from the mice revealed that mTOR KD was associated with decreased expression of Cdk6, a critical proliferative control node in T-cell development and oncogenic transformation. Pharmacologic inhibition of mTOR in tumor cells also decreased CDK6. The combination of a mTOR inhibitor (rapamycin) and a CDK4/6 inhibitor (PD-0332991, Palbociclib) synergistically decreased the overall viability and signaling downstream of drug targets in mouse lymphoma cells and in human T-ALL/LBL cell lines. This combination was also evaluated in mice using a disseminated leukemia model. In vivo treatment with this combination not only reduced tumor size by inhibiting tumor cell proliferation and arresting tumor cell cycle, but also increased overall survival. We are currently validating upstream regulators of Cdk6 as well as downstream targets in the pre-T LBL tumors from the mTOR deficient mice. Citation Format: Shuling Zhang, Joy M. Gary, John K. Simmons, Jinfei Xu, Benjamin J. Gamache, Ke Zhang, Nicholas Watson, Alexander L. Kovalchuk, Aleksandra M. Michalowski, Jin-Qiu Chen, Michelle A. Herrmann, Tuddow Thaiwong, Matti Kiupel, Wendy Dubois, Joseph R. Testa, Beverly A. Mock. Genetic and pharmacologic inhibition of mTOR delays mortality due to thymc lymphoma formation in mice and is associated with decreases in cell cycle proteins. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2833.

Objective In mast cell (MC) neoplasms, clinical problems requiring therapy include local aggressive and sometimes devastating growth of MCs and mediator-related symptoms. A key mediator of MCs responsible for clinical symptoms is histamine. Therefore, use of histamine receptor (HR) antagonists is an established approach to block histamine effects in these patients. Materials and Methods We screened for additional beneficial effects of HR antagonists and asked whether any of these agents would also exert growth-inhibitory effects on primary neoplastic MCs, the human MC line HMC-1, and on two canine MC lines, C2 and NI-1. Results We found that the HR1 antagonists terfenadine and loratadine suppress spontaneous growth of HMC-1, C2, and NI-1 cells, as well as growth of primary neoplastic MCs in all donors tested (human patients, n = 5; canine patients, n = 8). The effects of both drugs were found to be dose-dependent (IC50: terfenadine, 1–20 μM; loratadine, 10−50 μM). Both agents also produced apoptosis in neoplastic MCs and augmented apoptosis-inducing effects of two KIT-targeting drugs, PKC412 and dasatinib. The other HR1 antagonists (fexofenadine, diphenhydramine) and HR2 antagonists (famotidine, cimetidine, ranitidine) tested did not exert substantial growth-inhibitory effects on neoplastic MCs. None of the histamine receptor blockers were found to modulate cell-cycle progression in neoplastic MCs. Conclusions The HR1 antagonists terfenadine and loratadine, in addition to their antimediator activity, exert in vitro growth-inhibitory effects on neoplastic MCs. Whether these drugs (terfenadine) alone, or in combination with KIT inhibitors, can also affect in vivo neoplastic MC growth remains to be determined.

Systemic mastocytosis (SM) in felines is a rare neoplasm defined by increased growth and accumulation of immature mast cells (MC) in various organs including the spleen. Although in many cases splenectomy is an effective approach, relapses may occur. In these patients, treatment options are limited. Recent data suggest that various Kit tyrosine kinase inhibitors (TKI) interfere with growth of neoplastic MC in humans. In the current study, we examined the effects of four TKI, imatinib, midostaurin, nilotinib, and dasatinib, on growth of spleen-derived feline neoplastic MC in three SM patients. Expression of Kit in neoplastic MC was confirmed by flow cytometry and/or Western blotting. In all three cases, a 12-bp internal tandem duplication in exon 8, resulting in a four amino acid-insertion between residues Thr418 and His419 in Kit, was detectable. As assessed by (3)H-thymidine incorporation experiments, all four TKI were found to inhibit the growth of feline neoplastic MC in a dose-dependent manner. The growth-inhibitory TKI effects were found to be associated with morphologic signs of apoptosis in MC. In conclusion, various Kit-targeting TKI can inhibit the in vitro growth and survival of feline neoplastic MC in SM.

Background Breast cancer is one of the leading causes of cancer deaths. Triple-negative breast cancer (TNBC), an immunophenotype defined by the absence of immunolabeling for estrogen receptor (ER), progesterone receptor (PR) and HER2 protein, has a highly aggressive behavior. A subpopulation of TNBCs exhibit a basal-like morphology with immunohistochemical positivity for cytokeratins 5/6 (CK5/6) and/or epidermal growth factor receptor (EGFR), and have a high incidence of BRCA (breast cancer susceptibility) mutations. Feline mammary adenocarcinomas (FMAs) are highly malignant and share a similar basal-like subtype. The purpose of this study was to classify FMAs according to the current human classification of breast cancer that includes evaluation of ER, PR and HER2 status and expression of basal CK 5/6 and EGFR. Furthermore, we selected triple negative, basal-like FMAs to screen for BRCA mutations similar to those described in human TNBC. Methods Twenty four FMAs were classified according to the current human histologic breast cancer classification including immunohistochemistry (IHC) for ER, PR HER2, CK5/6 and EGFR. Genetic alteration and loss of heterozygosity of BRCA1 and BRCA2 genes were analyzed in triple negative, basal-like FMAs. Results IHC for ER, PR and HER2 identified 14 of the 24 (58%) FMAs as a triple negative. Furthermore, 11of these 14 (79%) triple negative FMAs had a basal-like subtype. However, no genetic abnormalities were detected in BRCA1 and BRCA2 by direct sequencing and loss of heterozygosity analysis. Conclusion FMAs are highly aggressive neoplasms that are commonly triple negative and exhibit a basal-like morphology. This is similar to human TNBC that are also commonly classified as a basal-like subtype. While sequencing of a select number of triple negative, basal-like FMAs and testing for loss of heterozygosity of BRCA1 and BRCA2 did not identify mutations similar to those described in human TNBC, further in-depth evaluation is required to elucidate a potential role of BRCA in the tumorigenesis of triple negative, basal-like FMAs. The strong similarities in clinical behavior, morphology and IHC phenotype suggest that triple negative, basal-like FMAs may be a suitable spontaneous animal model for studying novel therapeutic approaches against human basal-like TNBC.

The type III membrane tyrosine kinase receptor c-KIT is a proto-oncogene and has been shown to harbor activating mutations in a variety of cancers in humans and dogs. Specifically, deletion in codon 556-558, changing VQWKVV to VQFV (Del2) and deletion in codon 556-557, changing VQWKVV to VQVV (Del9) have been observed in both human and canine human gastrointestinal stromal tumors (GISTs). These and similar deletions are also found in some canine mast cell tumors (MCTs). In addition, internal tandem repeat duplications in exon 11 are also observed in canine MCTs. However, the significance of these mutations in driving the tumorigenic process has not been previously assessed. To directly test the hypothesis that mutant c-KIT isoforms observed in spontaneous tumors in humans and dogs are capable of driving the tumorigenic process, each isoform was ectopically expressed in canine primary fibroblasts which normally do not express c-KIT. Transfection of cells with constructs each expressing either the two exon 11 deletion mutants (Del2 and Del9) and one exon 11 duplication mutant, but not normal c-KIT, resulted in neoplastic transformation in vitro. Each transformed cell line was evaluated for the tumorigenic phenotype via the ability to invade matrigel, grow on soft agar and grow in low serum. All transformed cells displayed an invasive tumorigenic phenotype in contrast to normal fibroblasts. In vivo tumorigencity of each transformed cell line was evaluated using subcutaneous injection of one hundred thousand or one million cells of each cell line at three sites each in NSG (NOD scid gamma) immunodeficient mice. All transformed cell lines resulted in tumors at the injection site regardless of the dose. Histologically, all appeared to be sarcomas, with some showing ostoid deposition, thereby warranting the diagnosis of osteosarcoma. With Del2 transformed cells, five of the six tumors were osteosarcomas with two showing metastatic lesions upon necropsy. With Del9 transformed cells three out of six tumors were osteosarcomas and no metastasis was noted. With the duplication mutation three out of the six tumors were osteosarcoma. These findings provide strong support that these mutant c-KIT isoforms are sufficient to derive the tumorigenic process. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 13. doi:1538-7445.AM2012-13

Abstract 4936 Advanced mast cell (MC) disorders are characterized by uncontrolled growth of neoplastic MC in various organ systems, resistance to conventional cytoreductive drugs, and a poor prognosis. In most patients, transforming mutations in the KIT proto-oncogene are detectable and are considered to contribute to resistance. MC lines are an important model for analyzing drug resistance in neoplastic MC. We have established a novel canine mastocytoma cell line, NI-1 from a canine patient suffering from mast cell leukemia. NI-1 cells were found to harbour several homozygous KIT mutations including two single nucleotide mutations, one at nucleotide 107 (C to T, leading to a missense mutation, P36L) and one at nucleotide 1187 (A to G, leading to a missense mutation, Q396R), a 12 bp duplication at nucleotide 1263, and a 12 bp deletion at nucleotide 1550, the latter reflecting a transcriptional variant. NI-1 cells contained histamine and formed tumors in NOD-SCID IL-2Rgammanull (NSG) mice. As assessed by 3H-thymidine uptake, a number of targeted drugs were found to inhibit the proliferation of NI-1 cells at pharmacologically relevant concentrations. Among these drugs were the KIT kinase blockers midostaurin (IC50 2 μM). However, the HDAC inhibitor vorinostat was found to block growth of NI-1 cells (0.1-0.5 μM). Drug response profiling also revealed that NI-1 cells differ markedly from canine C2 mastocytoma cells harbouring an exon 11 KIT mutation, and the human mast cell lines HMC-1.1 (exon 11 mutation in KIT) and HMC-1.2 (exon 11 and exon 17 KIT mutations). In contrast to C2 cells, NI-1 cells were found to be largely resistant against the growth-inhibitory effects of masatinib, sorafenib, and sunitinib. By contrast, NI-1 cells were found to be even more sensitive against the growth-inhibitory effects of the mTOR blocker RAD001 and the PI3-kinase/mTOR inhibitor NVP-BEZ235. Together, our data suggest that certain KIT mutations may be associated with relative resistance of neoplastic MC against KIT-targeting drugs, a phenomenon that has also been described for human MC and the KIT mutant D816V that mediates resistance against masatinib and imatinib. The novel MC line NI-1 may serve as a novel tool to investigate the mechanisms of resistance against TKI in neoplastic MC. Disclosures: Valent: Novartis: Research Funding; Bristol-Myers Squibb: Research Funding.