Signalment:  

8-year-old female spayed German shepherd dog, Canis familiars. The dog developed acute weakness and vomiting. On clinical examination, the dog presented with pale mucous membranes, superficial breathing and abdominal distension. Abdominal radiographs revealed a moderate abdominal effusion. Cytology of abdominal fluid contained elevated numbers of red blood cells and rare mesothelial reactive cells, suggestive of hemoperitoneum. On exploratory laparotomy a primary splenic ruptured mass was observed and splenectomy was performed. The mass was diagnosed as hemagiosarcoma by histology. To prevent metastatic spread, post-operative chemotherapy with a combination protocol of doxorubicin and cyclophosphamide administered every 3 weeks for a total of 6 treatments was administered after suture removal. The cumulative dose of doxorubicin was lower than 240 mg/m². After the sixth treatment the dog was referred for sudden onset of general weakness. Heart failure with atrial fibrillation rapidly followed and the animal died spontaneously.

Gross Description:  

At necropsy severe dilation of left and right ventricles was observed. The myocardial wall was generally paler than normal with thin, white, longitudinal streaks beneath the epicardium. Mild pulmonary edema and hyperemia of major internal organs were present. The entire heart was collected for histopathology.

Histopathologic Description:

Sections from the ventricular free walls, interventricular septum, and left papillary muscle are provided. All samples provided have similar changes with variable severity: randomly distributed, degenerated cardiomyocytes are present single or in groups admixed with morphologically normal myocytes. Single cells or small groups of cardiomyocytes are characterized by intracytoplasmic, one to multiple, smooth-contoured, empty vacuoles ranging from 4 to 36 μm in diameter (vacuolar degeneration-Adria cells). In these cells, when visible, the nucleus is displaced to the periphery. Occasionally, scattered myocytes contain a perinuclear area of homogenous, pale eosinophilic cytoplasm, with loss of central cross striations (myofibril loss, myocytolysis). Scattered hypereosinophilic, angulated myocytes and fragmented myofibers are also evident. Cardiomyocytes are multifocally separated by mild interstitial edema and occasional microhemorrhages. Areas of myocardial fibrosis with minimal numbers of lymphocytes and plasma cells may be present in some sections. 

Morphologic Diagnosis:  

Moderate, multifocal chronic myocardial vacuolar degeneration and mild chronic interstitial edema and fibrosis.

Lab Results:  

Complete blood count, liver and renal panels and electrocardiography were performed before each treatment. All parameters were within normal limits for the entire duration of chemotherapy.

Condition:  

Adriamycin toxicity

Contributor Comment:  

Myocardial vacuolar degeneration (Adria cells), myocytolysis, interstitial edema and fibrosis are considered characteristic findings of doxorubicin-induced cardiomyopathy.(6,8) Doxorubicin (DOX) is known to be a dose-limiting cardiotoxic compound able to induce an irreversible dilated cardiomyopathy in several species including canine and human.(1,3,5) Congestive heart failure and death have been documented during the treatment but also months after the completion of chemotherapy in many species. Despite its toxicity, DOX is largely used alone or in combination in order to treat several neoplastic entities including hemangiosarcoma in dogs.(6) In humans, the incidence of DOX-induced cardiomyopathy is approximately 1.7% and the mortality rate once dilation is initiated reaches 50% of patients.(8)

Toxicity is related to the total cumulative dose administered, as well as to the acute peak concentration levels of the compound. Increased risk of DOX cardiotoxicity has also been correlated with young age, concurrent administration of additional chemotherapy compounds and viral diseases. Retrospective studies of human patients have revealed that more than 4% of patients who receive a cumulative dose of 500-550 mg per square meter of surface area develop congestive heart failure. The incidence rises to more than 18% over 551-600 mg/m².(8) Based on several clinical studies limiting the cumulative dose to less than 450 mg/m2 is the best line of defense against DOX cardiotoxicity. Alternative methods have been the concurrent administration of antioxidants, iron chelators, use of DOX analogues. However, none of the approaches have had major success. Recently, combined administration of the hematopoietic cytokines EPO, G-CSF and TPO has prevented DOX cardiomyopathy in animal models.(8) In this dog, a standard chemotherapy protocol for canine hemangiosarcoma with a cumulative dose of DOX lower than 240 mg/m² (recommended to minimize chronic cardiac toxicity) was used.(3) Myocardial vacuolization is described in early and chronic toxicity with a wide range of and from 122 to 265 mg/ m² body surface area (BSA) in clinical cases.(3) The overall cumulative dose causing fatal cardiomyopathy is therefore controversial, and dogs are generally considered more sensitive to cardiotoxic effects of doxorubicin than humans, where cardiomyopathy and congestive heart failure are reported to occur at a total dose greater than 550mg/ m² BSA.(2)

Prognosisis very poor in human patients that develop cardiomyopathy within four weeks after administration of DOX and the majority die within two weeks after onset of symptoms.(8) Among long survivors after DOX therapy, several develop heart failure six to ten years after conclusion of chemotherapy. The late-onset cardiotoxic effects of conventional anthracycline therapy highlight the need of lifelong monitoring the cardiac status in human patients. Several techniques (i.e. radionuclide angiography assessment of left ventricular ejection fraction, electrocardiography and echocardiographically derived ejection fraction) have been proven to be poor indicators of early changes and subclinical myocardial injury.(3,8) Serial endomyocardial biopsies are currently considered the most sensitive and specific indicators of doxorubicin-induced injury.(8) Biopsies are examined by electron microscopy and graded applying a semiquantitative scoring system(1) based on the percentage of myocytes affected by myofibrillar loss and cytoplasmic vacuolization. Ultramicroscopic markers including myofibril loss, retention of sarcoplasmic reticulum and cytoplasmic vacuolation are utilized to grade injury on a scale of 1 to 3; biopsy samples in which fewer than 5% of cells have typical changes are given a grade 1 while those with changes over 36% are graded 3, the highest severity.(8) This grading shows a linear correlation with left ventricular function determined by radionuclide angiocardiography and is helpful for clinical determination of continuation of DOX therapy.(8)

In veterinary medicine, no sensitive predictor tests are currently available to monitor patients treated with doxorubicin. Herman and co-workers (1981) observed that plasma enzymes CPK (creatine phosphokinase), LDH (Lactate Dehydrogenase) and SGOT (Serum Glutamic Oxaloacetic Transaminase) were not reliable indicators of slowly progressive cardiac damage. Similarly to human patients, sequential echocardiograms and EKGs (electrocardiogram) are not considered sensitive predictors for canine cardiomyopathy since no consistent correlation between the severity of rhythm disturbance and the pathologic myocardial changes have been observed.(2) Myocardial biopsies in dogs, although not routinely performed due to their invasiveness, have been experimentally proven to be a sensitive test to monitor the early doxorubicin-associated cardiotoxicity.(7)

Because DOX cardiotoxicity is dose dependent, it has been used to experimentally induce heart failure in different animal species such as dog, sheep, goats and rodents.(5) DOX is delivered by intravenous and intracoronary injections at small doses to induce heart failure without systemic toxicity. Experimental DOX heart failure develops via bilateral enlargement, ventricular wall thinning with decreased cardiac output. This model has been utilized to study several treatments for cardiac failure however the model has several limitations: the degree of left ventricular dysfunction varies, is characterized by high incidence of arrhythmias, high cost of multiple intracoronary injections and the irreversible and progressive heart damage.(5)

The mechanism of action of DOX and other antracycline compounds on tumor cells are still a matter of controversy. Suggested mechanisms are: intercalation into the DNA molecule leading to inhibition of transcription, generation of reactive oxygen species leading to lipid peroxidation and DNA damage, DNA binding and alkylation, DNA cross-linking, interference with DNA unwinding and helicase activity, inhibition of topoisomerase II and induction of apoptosis. 

Anthracycline compounds including DOX, produce reactive oxygen species (ROS) interacting with mitochondrial enzymes. ROS are produced in vitro by high concentration of DOX that binds to iron forming DOX-iron complexes that bind to DNA and induce production of partially reduced oxygen compounds. These radicals can damage DNA via strand break formation. However, high concentrations of DOX seem necessary and antioxidant compounds do not diminish DOX cytotoxicity.(8)

The mechanism of DOX-cardiomyopathy remains unclear but seems different from the one underlying DOXs anti-tumour activity. Most studies support the view that increase in oxidative stress evidenced by increases in ROS and lipid peroxidation play a key role along with reduction in antioxidant levels and sulfhydryl groups.(3,6) Other associated mechanisms proposed have been: inhibition of protein and DNA synthesis, lysosomal and mitochondrial changes, alteration of sarcolemmal Ca2+ transport, attenuation of adenylate cyclase, ATPase activities, imbalance in myocardial electrolytes and several others.(8) Also, DOX downregulates the expression of cardiac-specific genes including contractile proteins (alpha-actinin, myosin light and heavy chains, troponin I, desmin), and sarcoplasmic reticulum proteins. The reduction of myocardial contractility can be directly associated with reduction in muscle proteins. DOX additionally induces apoptosis of endothelial cells and cardiomycytes contrary to its cystostatic effect in tumor cells. This latest mechanism seems related to p53 activation. However, the role of apoptotic pathways in DOX cardiotoxicity is still controversial.(8)

Transcriptional profiling via genome wide transcriptome analysis has been utilized to determine early cardiac response to DOX in a rat model perfused with DOX leading only to mild cardiac dysfunction.(9) The main characteristics of cardiomyocyte reprogramming were the repression of transcripts involved in cardiac stress response and stress signaling, modulation of genes with cardiac remodeling capacity and upregulation of energy related pathways. This latest research supports the hypothesis that blunted response to stress and reduced danger signalling are prime components of DOX toxicity and can drive to cardiomyocyte damage.(9)

JPC Diagnosis:  

Heart, cardiomyocytes: Degeneration, multifocal, mild, with intracytoplasmic vacuolation and rare myocardial fibrosis. 

Conference Comment:  

The contributor provides an excellent review of doxorubicin cardiotoxicity. Conference participants discussed the severity of the lesions in this section, favoring the term mild to describe both the vacuolar degeneration and fibrosis. This spurred a discussion of the general correlation of clinical cardiac disease and severity of myocardial injury as observed histologically. This correlation is often poor, as a small lesion at a critical site within the heart can lead to death, while even more extensive lesions, if not affecting a critical area, are sometimes asymptomatic. Furthermore, it is not uncommon for animals that die peracutely from cardiac failure to show no detectable microscopic abnormalities.(5)

References:

1. Billingham ME, Bristow MR. Evaluation of anthracycline cardiotoxicity: predictive ability and functional correlation of endomyocardial biopsy. Cancer Treatment Symposia. 1984;3:71-76.
2. Gralla EJ, Fleischman RW, Luthra YK, Stadnicki SW. The dosing schedule dependent toxicities of adriamycin in beagle dogs and rhesus monkeys. Toxicology. 1979;13:263-73. 
3. Mauldin GE, Fox PR, Patnaik AK, Bond BR, Money SC, Matus RE. Doxorubicin-induced cardiotoxicosis. Clinical features in 32 dogs. J Vet Inter Med. 1992;6:82-88.
4. Miller LM, Van Vleet JF, Gal A. Cardiovascular system and lymphatic vessels. In: Zachary JF, McGavin MD, eds. Pathologic Basis of Disease. 5th ed. St Louis, MO: Elsevier Mosby; 2012:555.
5. Monnet E, Chachques JC. Animal models of heart failure: what is new? Ann Thorac Surg. 2005;79:1445-1453.
6. Ogilvie GK, Powers BE, Mallinckrodt CH, Withrow J. Surgery and doxorubicin in dogs with hemangiosarcoma. J Vet Intern Med, 1996;10:379-84.
7. Sparano BM, Gordon G, Hall C, Iatrapoulos MJ, Noble JF. Safety assessment of new anticancer compound, mitoxantrone, in Beagle dogs: comparison with doxorubicin. II. Histologic and ultrastructural pathology. Cancer Treat Rep. 1982;66:1145-1158.
8. Takemura G, Fujiwara H. Doxorubicin-induced cardiomyopathy from the cardiotoxic mechanisms to management. Prog Cardiovasc Dis. 2007;49:330-352.
9. Tokarska-Schlattner M, Lucchinetti E, Zaugg M, Kay L, Gratia S, Guzun R, et al. Early effects of doxorubicin in perfused heart: transcriptional profiling reveals inhibition of cellular stress response genes. Am J Physiol Regul Integr Comp Physiol. 2010;298: R1075-1088.

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3-1. Heart

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