ESVP-ECVP Case 1: Edouard Reyes-Gomez

ENVA, France

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CASE TITLE

An unusual bone lesion in a young dog

SIGNALMENT

7-month-old dog, German shepherd, male

HISTORY

2-month history of a weight-bearing lameness of the left forelimb, associated with a marked and rapidly expanding swelling of the left forearm. Radiographs showed a large, cystic and osteolytic lesion of the left ulnar diaphysis. The histologic specimen submitted by the surgeon corresponds to tissue at the interface between the remaining diaphysis and the lesion.

What is your diagnosis? Which additional informations / tests may be useful in this case?


ESVP-ECVP Case 2: Edouard Reyes-Gomez

ENVA, France

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Slide ID: 14 0147 31

CASE TITLE

A rare lesion in a young dog

SIGNALMENT

11-month-old dog, Griffon vendéen, male

HISTORY

The dog was presented for a 2-week-history of right exophtalmia. The referring vet extracted the 2nd right superior molar and diagnosed a teeth root abscess. Bacterial culture identified Enterobacter sp. After a transient improvement following antibiotherapy, the dog was presented to a referral center due to persistent and worsening exophtalmia and conjunctival hyperhemia. Following MRI, clinicians suspected a retrobulbar abscess. The histologic specimen submitted by the surgeon is a fragment from the retrobulbar tissue.

What is your diagnosis? Which additional informations / tests may be useful in this case?


ESTP Case 1: Giovanni Pellegrini

Laboratory for Animal Model Pathology, University of Veterinary Pathology, University of Zürich

CASE TITLE

Renal Lesions in Folate Receptor-Targeted Radionuclide Therapy

ABSTRACT

Targeted radionuclide therapy is based on the systemic application of radioligands with high affinity to antigens on the surface of tumour cells. Folate receptor (FR)-targeted therapies employ particle-emitting radiopharmaceuticals coupled to the vitamin folic acid, which binds with high affinity to the FR expressed in several epithelial tumours; however, undesired accumulation of radioactivity is observed also in the kidneys, where FR is expressed at high levels in the proximal tubule epithelial cells. This case presents an example of chronic radiation nephropathy in a mouse administered with a radiofolate and discusses the use of potential  biomarkers (plasma CRE, BUN, Kim-1; urinary cystatin C, Kim-1, NGAL, NAG and IL-18; and immunohistological analysis of DNA double strand breaks in the kidneys using an anti-gamma-H2AX antibody) for the early detection of murine radiation nephropathy and prediction of renal outcome.

ANIMAL(S)

Two mice: AM32 (control) and  BM41 (treated)

SPECIES, BREED

Atymic nude mice (Crl:CD1-Foxn1nu)

SEX

F

AGE

40 weeks

STUDY TYPE

Chronic renal toxicity study

TREATMENT

Single i.v. injection of saline (AM32) or 177Lu-folate (BM41) at 30 MBq (megabecquerel), euthanised 7 months post injection

CLINICAL FINDINGS

Rough hair coat, decreased motor activity, body weight loss (mouse BM41)

ORGAN(S)

Kidneys (AM32 next to the slide label, BM41 on the opposite side)

GROSS FINDING(S)

Small kidneys with irregular surface (mouse BM41)

MORPHOLOGIC DIAGNOSIS

Kidneys: Severe diffuse chronic bilateral tubular loss, with  tubulointerstitial nephritis, glomerulosclerosis, glomerular mesangial expansion, and nuclear enlargement.

DESCRIPTION AND COMMENTS

DESCRIPTION: All components of the nephron are diffusely affected. Glomerular changes range from reduced numbers of capillaries and mesangial cells to mesangiolysis and mesangiosclerosis, with obsolescence of the glomerular tuft. Tubular atrophy and tubulolysis occur in the cortex, along with tubular casts, leading to prominent cortical collapse. Karyomegaly is occasionally noted in the glomerular tuft and the tubular epithelium. Interstitial changes, dominated by fibrosis and mild mononuclear cell infiltration, are also multifocally observed.

COMMENTS: The morphological and functional changes resulting from the toxic effects of radiations on the kidneys  are referred to as radiation nephropathy, a chronic progressive, irreversible condition occurring in both humans and laboratory animals characterized by a long latency phase after exposure and then sudden onset of glomerular and tubular alterations leading eventually to end stage kidney. In laboratory animals, histological features consistent with radiation nephropathy are frequently observed only months after irradiation, while the early stages post exposure are dominated by subtle histological changes, variable among the different species and whose predictive value has not been studied yet. In mice and rats for example, the earliest microscopic findings detected in the kidneys following irradiation consist of tubulolysis and tubular reactive changes, which occur 4 or 6 weeks after irradiation, whilst, for the recognition of other features suggestive of radiation nephropathy,  a few (2-3) to several (10) months might be necessary.

LITERATURE

[1] Gudkov SV, Shilyagina NY, Vodeneev VA, and Zvyagin AV. Targeted radionuclide therapy of human tumors. International journal of molecular sciences 2015;17.

[2] Lambert B, Cybulla M, Weiner SM, Van De Wiele C, Ham H, Dierckx RA, et al. Renal toxicity after radionuclide therapy. Radiation research 2004;161:607-11.

[3] Cohen EP and Robbins ME. Radiation nephropathy. Seminars in nephrology 2003;23:486-99.

[4] Müller C and Schibli R. Prospects in folate receptor-targeted radionuclide therapy. Frontiers in oncology 2013;3:249.

[5] Haller S, Pellegrini G, Vermeulen C, van der Meulen NP, Köster U, Bernhardt P, et al. Contribution of Auger/conversion electrons to renal side effects after radionuclide therapy: preclinical comparison of 161Tb-folate and 177Lu-folate. EJNMMI research 2016;6:13.


ESTP Case 2: Stéphane Lezmi

S. Lezmi*, F. Sebal*, C. Favre-Guilmard†, D. Carre‡, V. Martin*, M. Kalinichev†, F. Schmidlin‡ and C. George*

*Non-Clinical Drug Safety, †Neurology and ‡Biomarker Departments, Ipsen innovation, France

CASE TITLE

Evaluation Of Botulinum Neurotoxin Type A Effects On The Distribution Of Cleaved-snap25 In Muscles And Spinal Cord Of Rats Injected In The Gastrocnemius Muscle Over A 75-day Period

ABSTRACT

Introduction: Botulinum neurotoxin type A (BoNT/A) is used for the treatment of muscle hypertonicity in various medical indications, and induces a long-lasting muscle relaxation by cleaving the synaptic protein SNAP25. Little is known about the distribution of BoNT/A after a single intramuscular injection.

Materials and Methods: Twenty-four rats received 125 pg/kg of natural BoNT/A (List) in the left gastrocnemius (highest tolerated dose without body weight effect in this species). The digit abduction score (DAS) was evaluated, and rats sacrificed 1 hour post administration and at different timepoints up to Day 75. Muscles and spinal cord were sampled for morphological analyses and detection of cleaved-SNAP25 (C-SNAP25) by immunohistochemistry.

Results: Treated rats presented with a maximum DAS on D3 and 6, which progressively decreased up to D27, suggesting a diffusion of the toxin from the gastrocnemius to the extensor digit muscles in which levels of C-SNAP25 strongly correlated with the digit abduction inhibition. In the injected gastrocnemius, high levels of C-SNAP25 were gradually detected at neuromuscular junctions from H1 up to D27, and progressively decreased. The distribution of C-SNAP25 and morphological changes were also evaluated in multiple muscles. In the lumbar spinal cord, a strong C-SNAP25 staining was progressively observed from D1 to up to D27 and gradually decreased.

Conclusions: The detection of C-SNAP25 appears to closely reflect the action of BoNT/A in muscles at early stages. However, it’s detection at later stages may not reflect the direct activity of BoNT/A, but rather a persistence of the cleaved protein in various tissues.


ESTP Case 3: Junwen Qiao and Jin Ren

Center for Drug Safety Evaluation and Research, Shanghai Institute of Material Medica,China Academy of Science

CASE TITLE

IL-15 Superagonist Induced Lymphocytic Hyperplasia and Lymphocytic Infiltration

ABSTRACT

Interleukin-15 (IL-15) is a four-helix common γ-chain cytokine that plays a crucial role in the development, maturation, and activation of NK cells and CD8+ memory T cells.  Administration of exogenous IL-15 facilitates the expansion of NK and CD8+ T cell populations, both of which play important roles in anticancer and antiviral immunosurveillance.  Recent research has found that a combination of IL-15 and the IL-15Ra subunit, termed IL-15 superagonist (IL-15 SA), produced a compound with significantly greater biological activity than naive IL-15. IL-15 SA might serve as an excellent immunotherapeutic candidate for the treatment of cancer; several similar compounds are currently in clinical trials or a preclinical safety phase. To our knowledge, however, there are only limited data available on the preclinical histopathology produced by this type of compound.

In one of our preclinical 4-week toxicity study, administration of IL-15 SA to SD rats (once weekly) by intravenous injection resulted in mortality at the high dose level.  IL-15 SA administration caused gross findings of enlarged spleen, lymph nodes and liver; organ weight increases in the spleen and liver were noted at high and middle doses.  Microscopically, IL-15 SA administration resulted in substantial lymphocytic hyperplasia in the spleen and lymph nodes, as well as lymphocytic infiltration in multiple organs, primarily the liver, lung and adrenal glands.  Lymphocytic hyperplasia in the spleen was characterized by proliferation of lymphocytes primarily in the red pulp with expansion through the capsule. Lymphocytic hyperplasia in the lymph nodes were most apparent in the paracortex, and to a lesser degree in the medulla often resulting in indistinct cortex-medulla demarcation.  Lymphocytic infiltration in the liver was characterized by presence of substantial numbers of lymphocytes around the central veins, periportal blood vessels and/or in the sinusoid. Scattered foci of hepatocellular necrosis and hypertrophy of bile ducts were also observed. The lung had a perivascular/peribronchiolar pattern of lymphocyte infiltration with occasional foci of alveolar macrophage aggregates and inflammation.  Lymphocytic infiltration in other organs generally had lower incidence and severity grades and was mainly present in the nonparenchymal components such as capsules and connective tissues.  Additional affected organs included aorta, thymus, stomach, ileum, ovaries, oviducts, vagina, uterus, cervix, kidneys, urinary bladder, pancreas and knee joint.  All of those induced changes were completely resolved or showed recovery trend following 4-week recovery.   Identification of IL-15 SA-induced histopathology is important in the accurate safety assessment and development of these compounds.

ANIMAL(S)

Rat

SPECIES, BREED

Sprague Dawley

SEX

Male and Female

AGE

11-12 Weeks

STUDY TYPE

4-Week Intravenous Injection

TREATMENT

Once Weekly

CLINICAL FINDINGS

--

ORGAN(S)

Spleen, Lymph node, Liver, Lung and Adrenal glands

GROSS FINDING(S)

Enlarged spleen, liver and lymph nodes.

MORPHOLOGIC DIAGNOSIS

Lympohocytic hyperplasia in the spleen and lymph node; lymphocytic infiltration in the liver, lung, adrenal glands and other organs.

DESCRIPTION AND COMMENTS

IL-15 SA administration resulted in substantial lymphocytic hyperplasia in the spleen and lymph nodes, as well as lymphocytic infiltration in multiple organs, primarily the liver, lung and adrenal glands. Lymphocytic hyperplasia in the spleen was characterized by proliferation of lymphocytes primarily in the red pulp with expansion through the capsule.  Lymphocytic hyperplasia in the lymph nodes were most apparent in the paracortex, and to a lesser degree in the medulla often resulting in indistinct cortex-medulla demarcation.  Lymphocytic infiltration in the liver was characterized by presence of substantial numbers of lymphocytes around the central veins, periportal blood vessels and/or in the sinusoid. Scattered foci of hepatocellular necrosis and hypertrophy of bile ducts were also observed.  The lung had a perivascular/peribronchiolar pattern of lymphocyte infiltration with occasional foci of alveolar macrophage aggregates and inflammation.  Lymphocytic infiltration in other organs generally had lower incidence and severity grades and was mainly present in the nonparenchymal components such as capsules and connective tissues. Additional affected organs included aorta, thymus, stomach, ileum, ovaries, oviducts, vagina, uterus, cervix, kidneys, urinary bladder, pancreas and knee joint. 

LITERATURE

Guo Y, Luan L, Rabacal W, Bohannon JK: IL-15 Superagonist-Mediated Immunotoxicity: Role of NK Cells and IFN-γ. J Immunol. 2015 Sep 1;195(5):2353-64


ESTP Case 4: Torrie A. Crabbs

Experimental Pathology Laboratories, Inc., Research Triangle Park, Raleigh, North Carolina, USA

CASE TITLE

When "Liver" Lesions Appear in the Pancreas: A Diagnostic Challenge

ABSTRACT

In rats, induction of cholangiofibrosis has been documented following administration of several compounds, most notably furan. However, it has not been observed in untreated control F344 rats in National Toxicology Program studies. In fact, the only rat strain in which cholangiofibrosis has been reported to occur in non-treated animals is the Long-Evans Cinnamon (LEC) rat - a rat model of Wilson's disease that is characterized by a ceruloplasmin deficiency, hepatic copper accumulation, and hepatocellular injury. This case presents 2 examples of spontaneous, non-exposure related cholangiofibrosis in aged female Harlan Sprague Dawley rats from an ongoing carcinogenicity/chronic toxicity study. While the histomorphologic features are identical to those of induced cholangiofibrosis following furan administration, its location/distribution (extra-hepatic, periductal) and incidence (non-treatment related) are unique. 

ANIMAL(S)

Rat

SPECIES, BREED

Harlan Sprague Dawley (HSD) rats

SEX

Female

AGE

~2 yrs

STUDY TYPE

Carcinogenicity/Chronic Toxicity Study

TREATMENT

Compound still on study

CLINICAL FINDINGS

None

ORGAN(S)

Pancreas

GROSS FINDING(S)

None

MORPHOLOGIC DIAGNOSIS

Pancreas, periductal - cholangiofibrosis

DESCRIPTION AND COMMENTS

This lesion is within the region of the pancreas adjacent and/or immediately proximal to the entrance of the common bile duct into the proximal duodenum. The lesion is characterized by ducts surrounded by abundant dense, collagenous connective tissue. The ducts are  are often distorted, tortuous, and distended by variable, but often copious, amounts of pale basophilic to eosinophilic fibrillary to amorphous mucin-like material, in addition to sloughed lining cells, and/or necrotic debris. The lining epithelium is cuboidal to columnar, exhibits occasional crowding or multi-layering, and contains scattered to abundant goblet cells (intestinal metaplasia). Variable numbers of mixed inflammatory cells were often scattered through the fibrous stroma.

LITERATURE

Adams EA, Auerbach S, Blackshear PE, Bradley A, Gruebbel MM, Little PB, Malarkey D, Maronpot RR, McKay JS, Miller RA, Moore RR, Morrison JP, Nyska A, Ramot Y, Rao D, Suttie A, Wells MY, Willson GA, Elmore SA (2011). Proceedings of the 2010 National Toxicology Program Satellite Symposium. Toxicol Pathol 39: 240 – 266.

Eustis SL, Boorman GA, Harada T, Popp JA (1990). Liver. In: Pathology of the Fischer Rat: Reference and Atlas, eds. GA Boorman, SL Eustis, MR Elwell, CA, Montgomery, WF MacKenzie, Academic Press, San Diego, pp. 71 – 94.

Thoolen B, Maronpot RR, Harada T, Nyska A, Rousseaux C, Nolte T, Malarkey DE, Kaufmann W, Küttler, Deschl U, Nakae D, Gregson R, Vinlove MP, Brix AE, Singh B, Belpoggi F, Ward JM (2010). Proliferative and nonproliferative lesions of the rat and mouse hepatobiliary system. Toxicol Pathol 38: 5S – 81S.

ADDITIONAL COMMENTS

The presented cases are from an on-going study in which focal cholangiofibrosis was noted in eight male and twenty-one female HSD rats; two affected animals (one male and one female) were unexposed controls. Findings were considered incidental and unrelated to exposure. In males, 100% of cases (8/8) were located in or closely associated with the liver; whereas 76% of cases in females (16/21) were associated with the pancreas, as presented. To the authors’ knowledge, this is the first report of cholangiofibrosis as a spontaneous change in rats and also the first report of cholangiofibrosis in an extrahepatic location (pancreas).


ESTP Case 5: Danielle Brown

Charles River Laboratories, Durham, NC, USA

CASE TITLE

Use of Stereology in Animal Model Development and Efficacy Evaluation

ABSTRACT

The development of applicable animal models of human disease is a necessary aspect of translational research and compound efficacy studies.  Although 2-dimensional morphometric analysis can be of use to characterize and evaluate these models, the methods used lack standardization and are often highly biased due to multiple assumptions about the tissue and a lack of unbiased sampling paradigms.  Thus, the results may not be accurate and the methods may not be sensitive enough to detect subtle differences between treatment groups once the model has been established.  Previous studies have shown that results obtained by 2-dimensional methods can even be in the opposite direction of the truth.1  Because of this, several professional societies and high-impact journals have published position papers on the need for unbiased quantitative methods, particularly for certain tissues such as the lung and brain.2,3,4,5

Stereology is a design-based technique based on stochastic geometry and statistical principles that does not make assumptions about the tissue and is therefore considered unbiased.6  Standardized sampling and counting methods can be performed on virtually any tissue from any species, and the results obtained are absolute 3-dimensional estimates rather than densities or ratios.  Because stereology is an unbiased, design-based method, it has the sensitivity required to detect subtle differences between groups and is thus highly applicable to animal model development and evaluation of compound efficacy within these models. 

The objective of this presentation is to present the basic principles and applications of stereology and how they can be applied to animal model development and evaluation, including several real-life examples.  Time for discussion and interactive audience participation will be included at the end of the presentation.

REFERENCES

  1. Boyce RW, Ebert DC, Youngs TA, Paddock CL, Mosekilde L, Stevens ML, Gundersen HJG. Unbiased estimation of vertebral trabecular connectivity in calcium-restricted ovariectomized minipigs. Bone. 1995;16:637-642.
  2. Hsia CCW, Hyde DM, Ochs M, Weibel ER. An official research policy statement of the American Thoracic Society / European Respiratory Society: standards for quantitative assessment of lung structure. Am J Respir Crit Care Med. 2010;181:394-418.
  3. Coggeshall RE, Lekan HA. Methods for determining numbers of cells and synapses: a case for more uniform standards of review. J Compar Neurol. 1996;364:6-15.
  4. Saper CB. Any way you cut it: a new journal policy for the use of unbiased counting methods. J Compar Neurol. 1996;364:5.
  5. West WJ, Coleman PD. How to count. Neurobiol Aging. 1996;17:503.
  6. Sterio DC. The unbiased estimation of number and sizes of arbitrary particles using the disector. J Microsc. 1984;134:127-136.