Toxicologic Pathology Forum: Opinion on Obligatory Microscopic Examination of Intermediate-Dose Groups in Toxicity Studies With Biotherapeutics in Cynomolgus Monkeys.

In nonrodent toxicity studies that are usually conducted in cynomolgus monkeys or beagle dogs, the added value of examining all tissues from all dose groups (current practice) versus all tissues in only control and high-dose groups and target tissues in intermediate-dose groups by default, is a subject of debate. A previous retrospective review of 325 nonrodent toxicity studies that included a limited number of biotherapeutics suggested that the evaluation of all tissues from all groups was not justified as a routine practice and recommended the examination of all tissues in control and high-dose groups and only target tissues in intermediate-dose groups. In contrast, the present retrospective review which examined 213 nonrodent studies (212 in cynomolgus monkeys and 1 in dog) from 4 multinational pharmaceutical companies (Bristol-Myers Squibb, Novartis, Pfizer Inc, and Roche) conducted only with biotherapeutics showed that restricting the microscopic examination in intermediate-dose groups to target tissues has the potential to miss findings in 6.6% of studies, possibly impacting the overall study interpretation and conclusion. In conclusion and in the opinion of the authors, all tissues from all dose groups should be examined in toxicity studies with biotherapeutics conducted in nonrodent species.

Evaluation of microRNAs-208 and 133a/b as differential biomarkers of acute cardiac and skeletal muscle toxicity in rats.

Conventional circulating biomarkers of cardiac and skeletal muscle (SKM) toxicity lack specificity and/or have a short half-life. MicroRNAs (miRNAs) are currently being assessed as biomarkers of tissue injury based on their long half-life in blood and selective expression in certain tissues. To assess the utility of miRNAs as biomarkers of cardiac and SKM injury, male Sprague-Dawley rats received a single dose of isoproterenol (ISO); metaproterenol (MET); allylamine (AAM); mitoxantrone (MIT); acetaminophen (APAP) or vehicle. Blood and tissues were collected from rats in each group at 4, 24 and 48h. ISO, MET, and AAM induced cardiac and SKM lesions and APAP induced liver specific lesions. There was no evidence of tissue injury with MIT by histopathology. Serum levels of candidate miRNAs were compared to conventional serum biomarkers of SKM/cardiac toxicity. Increases in heart specific miR-208 only occurred in rats with cardiac lesions alone and were increased for a longer duration than cardiac troponin and FABP3 (cardiac biomarkers). ISO, MET and AAM induced increases in MyL3 and skeletal muscle troponin (sTnl) (SKM biomarkers). MIT induced large increases in sTnl indicative of SKM toxicity, but sTnl levels were also increased in APAP-treated rats that lacked SKM toxicity. Serum levels of miR-133a/b (enriched in cardiac and SKM) increased following ISO, MET, AAM and MIT treatments but were absent in APAP-treated rats. Our results suggest that miR-133a/b are sensitive and specific markers of SKM and cardiac toxicity and that miR-208 used in combination with miR-133a/b can be used to differentiate cardiac from SKM toxicity.

Severe Acquired Idiopathic Thrombocytopenia in a Female Cynomolgus Macaque (Macaca fascicularis).

A 4-y-old female cynomolgus macaque presented for veterinary evaluation prior to placement in a preclinical study showed markedly low platelet counts that continued to decrease over time. Physical examination over the next several days showed areas of pale red discoloration in forelimbs, anterior thorax, and inguinal area and multifocal pinpoint areas of erythema or scabs. An area of dark red discoloration approximately 2 cm in diameter on the dorsal surface of the tongue was discovered on day 9. The macaque was euthanized, and histopathologic evaluation showed multifocal, ulcerative or erosive, hemorrhagic, lymphohistiocytic and neutrophilic glossitis and tonsillitis. The lesions on the tongue were associated with opportunistic fungi consistent with Candida albicans. The bone marrow showed megakaryocytic hyperplasia. There was no evidence of increased consumption of platelets, sequestration of platelets, or bone marrow suppression. The monkey was serologically negative for simian retrovirus, SIV, and simian T-lymphotropic virus. In light of cases reported in humans, immune-mediated destruction of platelets due to autoantibodies secondary to Candida albicans infection was considered. However, we were unable to detect antiplatelet antibodies on the platelet surface or in serum to support this etiology; therefore idiopathic thrombocytopenia was diagnosed. To our knowledge, this case represents the second reported observation of acquired thrombocytopenia in a nonhuman primate and the first reported observation in a cynomolgus macaque.

Measles virotherapy in a mouse model of adult T-cell leukaemia/lymphoma.

Adult T-cell leukaemia/lymphoma (ATL) is a highly aggressive CD4(+) T-cell malignancy caused by human T-cell leukaemia virus type 1. Measles virus (MV) oncolytic therapy has been reported to be efficient in reducing tumour burden in subcutaneous xenograft models of lymphoproliferative disorders such as myeloma, B-cell lymphoma and cutaneous T-cell lymphoma, but its potential to reduce tumour burden in disseminated lymphoproliferative disorders such as ATL remains to be determined. In this study, MV oncolytic therapy was evaluated in the MET-1/NOD/SCID xenograft mouse model of ATL. Treatment with the vaccine-related strain MV-NSE led to a significant reduction in tumour burden. In mice with a high tumour burden, therapy with MV-NSE significantly increased survival beyond any other single treatment tested previously using this model. Interestingly, signs of morbidity (cachexia) in mice treated with MV were not directly associated with tumour burden, but were correlated with the secretion of interleukin-6 by MET-1 cells and host cells. The results suggest that MV therapy could be a promising therapy for generalized lymphoproliferative disease.

Multicentric mast cell tumors in a horse.

A 6-year-old female Rocky Mountain horse was presented for evaluation of draining tracts and distal limb subcutaneous edema on the left front and left hind limbs that had been present for 2 weeks. Direct smears of fluid collected by fine-needle aspiration of subcutaneous fluid from both limbs were highly cellular with a predominance of eosinophils accompanied by numerous, moderately atypical, variably granulated mast cells. The cytologic diagnosis was mast cell tumor (MCT) with prominent eosinophilic infiltration with a differential diagnosis of eosinophilic granuloma. Histologic evaluation of surgical biopsies of lesions from both limbs was performed on sections stained with H&E, toluidine blue, and Luna stains. The histologic diagnosis was MCT, and staining with toluidine blue and Luna stains confirmed the presence of mast cells and eosinophils, respectively. In addition, the mast cells strongly expressed CD117. This is the first reported case of cutaneous mast cell neoplasia in a horse in which primary presenting complaints were draining tracts and distal limb subcutaneous edema involving multiple limbs. This case illustrates the utility of staining for CD117 expression in combination with traditional stains, such as toluidine blue and Luna, in differentiating MCTs from other eosinophilic lesions in horses.

Cervical thymoma originating in ectopic thymic tissue in a cat.

An 11-year-old female spayed domestic shorthair cat was referred to The Ohio State University Veterinary Teaching Hospital (OSU-VTH) for evaluation of a 6 x 4 x 3.5 cm mass in the left midcervical region causing increased respiratory sounds and lateral deviation of the trachea. A fine needle aspirate of the mass was obtained before referral and the cytology results were compatible with a reactive lymph node. Immunocytochemistry showed increased numbers of CD3+ T lymphocytes and small numbers of CD20+ and CD79a+ medium to large lymphocytes. Differential diagnoses from the referral pathologist were T-cell-rich B-cell lymphoma and feline Hodgkin's-like lymphoma. A subsequent fine needle aspirate performed at the OSU-VTH showed similar results. On flow cytometry the majority of cells were CD3+ T lymphocytes that were double positive for CD4 and CD8 (73%), compatible with either a double-positive (CD4+CD8+) T-cell lymphoma or lymphocytes from ectopic thymic tissue. The mass was surgically removed. Histopathology and immunohistochemistry of the mass revealed a predominant population of CD3+ small lymphocytes and small numbers of medium to large lymphocytes with moderate anisocytosis and anysokaryosis. A population of cytokeratin-positive epithelial cells surrounded small microcystic structures filled with eosinophilic material and structures interpreted as Hassall's corpuscles. These findings were consistent with thymic tissue and a diagnosis of ectopic thymoma was made. PCR results for lymphocyte antigen receptor rearrangement (PARR) were negative. The cat had no evidence of disease 16 months after removal of the mass. To our knowledge this is the first report of an ectopic cervical thymoma in a cat. The clinical and diagnostic features of this unusual case will be useful in helping veterinarians and pathologists obtain a presurgical diagnosis and establish a prognosis for similar lesions.