Ultrasonography of Osteosarcoma in Dogs
Sonography Atlas of Dogs

Purpose. The feasibility of hyperthermic isolated limb perfusion (HILP) with tumor necrosis factor-α (TNFα ) and cisplatin for the management of osteosarcoma was studied in the canine model.

Methods. During seven perfusions in six healthy mongrel dogs (weight 32±2 kg) technical aspects of HILP under mild hyperthermia (39– 40℃) were studied. In five experiments HILP was performed with TNFα alone (0.5 mg/l extremity volume), and in two experiments TNFα was combined with cisplatin (25 mg/l extremity volume). During the perfusions physiological parameters were monitored and TNFα and total cisplatin concentrations were determined.

Results. Perfusion conditions (pH, PCO₂ , PO₂, flow and pressure) remained within physiological ranges.Three dogs died within 24 h despite a sublethal systemic concentration of TNFα that leaked from the perfusion circuit. Three dogs were terminated; one dog after the second experiment in accordance with Dutch ethical rules; one dog showed an invagination of the small bowel resulting in an ileus; one dog because of necrosis of the perfused limb.

Conclusions. This feasibility study in healthy dogs demonstrated that HILP with TNFα and cisplatin was associated with a high mortality rate and does not allow us to treat dogs with spontaneous osteosarcoma with TNFα and cisplatin HILP. Therefore, an alternative model should be used in the search for the ideal combination of perfusion agents for limb sparing treatment in human osteosarcoma.

 

Introduction
Osteosarcoma is the most frequent primary malignant bone tumor in humans. Until the 1970s the most
common approach to the management of localized osteosarcoma was surgical resection, amputation or
radiation therapy.1 During the last decades a definite role for neoadjuvant high-dose methotrexate and
cisplatin-based polychemotherapy was established.1-4 The potential local tumor effect of systemically
administered cisplatin, however, is limited due to the nephrotoxicity and ototoxicity of cisplatin. Therefore
an attempt was made to increase the local effe ct of cisplatin without increasing systemic toxicity by using
hyper thermic isolated regional limb perfusion (HILP) with cisplatin in dogs with spontaneous osteosar-coma.5
These studies showed an acceptable locore-gional toxicity, improved functional outcome at 6
and 12 weeks, and a steadily improving radiological picture. However, the histological results were modest,
with none of the dogs showing a complete response at 6 weeks after perfusion. The same experience was
found in patients with sarcomas of soft tissue and bone treated with cisplatin HILP.6
Results of recent publications and of our own experience with a new perfusion modality, which combines tumor necrosis factor-a(TNFa) and melphalan in patients with recurrent melanoma or soft tissue sarcoma, are very promising. 7,8 However, in six of eight evaluable patients with unresectable osteosarcoma of the lower limb treated wi th TNFaand melphal an HILP, histological evaluation revealed moderate results with > 80% necrosis in three patients, 50-60% necrosis in two patients and < 50% necrosis in one patient. After TNFaand melphalan HILP, limb spar ing surgery was possible in six patients.9 As cisplatin is one of the most active chemotherapeutics in the treatment of osteosarcoma, it seems worthwhile to investigate the high frequency high frequency of occurrence in dogs, canine osteosarcoma is a useful model for evaluation of new treatment regimens in humans as rapid case accrual and rapid time to reach measurabl e end poi nts are possible.10 The canine osteosarcoma therefore appears
to be a valid model for studying the potential treat-ment of HILP with TNFaand cisplatin in the local treatment of osteosarcoma of the extremity in humans. To establish optimal HILP conditions using TNFaand cisplatin for local tumor control in dogs bearing osteosarcoma, a feasibility study in healthy dogs was undertaken.

Materials and methods
Dogs
During seven experiments in six healthy mongrel dogs with a mean average weight of 32 ± 2 kg and a mean
age of 6 ± 1 years, different aspects of HILP with TNFaand cisplatin were studied. Preoperatively, all
dogs were thoroughly clinically evaluated at the Central Animal Facility of the University of Groningen.
The study was approved by the Animal Welfare Committee of the Faculty of Medicine of the Gron-ingen
University.

Anaesthetics
The dogs fastened for 12 h and were anaesthetized with thiopental (30 mg/kg body wt., i.v.) (Pentothal,
Abbott, Amstelveen, The Netherlands) and, after musclerelaxation with pancuroni umbromide
(0.08 mg/kg body wt., i.v.) (Pavulon, Organon, Oss, The Netherlands), the dogs were ventilated (Ohmeda
Modulus 2) with a mixture of O2 and isoflurane. The oxygen concentration in the gas mixture was continuously
measured by means of an oxygen analyzer (Ohmeda Modulus 2) and minute volumes (4±6 l/ min) were adjusted to maintain an end-expiratory CO 2 concentration of 4-5% (Siemens CO 2 analyzer 930). The dogs were placed in the supine position on a heated mattress to maintain their normal body temperature of 38°C.11 During the operations all
dogs were given about 2 l of glucose 5% via acephalicor internal jugular vein. Central arterial pressure was
recorded as well as an ECG and diuresis.

 

Operation and perfusion techniques

During anaesthesia the volume of the extremity was measured using Archimedes’ rule (1.7±2 l). The iliac vessels were exposed under sterile conditions and collateral vessels were clipped. Cannulas were inserted into the artery (Bardic, 14-18 F) and vein (Bardic, 14-18 F). Both cannulas were connected to an extracorporeal circuit consisting of an occlusive roller pump, a cardiotomy reservoir and a bubble oxygenator with heat-exchanger. A tourniquet made of nylon was placed around the base of the extremity, using a pin in the bone and a bandage around the middle to complete the isolation of the limb from the systemic circulation. The perfusate consisted of 350 ml 5% dextran 40 in glucose 5% (Isodex, Phar macia AB, Uppsala, Sweden), 250 ml red blood cells (canine blood donor s), 250 ml plasma, 30 ml sodium bicarbonate 8.4% and 0.5 ml 5000 IU/ml heparin ( Thromboliquine, Organon B. V. , Oss, The Netherlands). The mixture of oxygen, air and carbon dioxide through the oxygenator was adjusted to maintain the blood gas values within the physiological range and, when necessar y, bicarbonate was added to adjust the pH value. All perfusions were performed under mild hyperthermic conditions (39-40°C) and optimal physiological conditions.12,13 Thermistor probes ( Electrolaboriet , Copenhagen, Denmark) were inserted into the subcutaneous tissues and into a muscle of the thigh just above the knee for continuous monitoring of the temperatures during perfusion. In the first five experiments TNFawas the sole perfusion agent, i n the l ast t wo experi ments TNFawas
combi ned with cispl atin. The dosage of TNFa (0.5 mg/l extremity volume) (Boehringer, Ingelheim,
Germany) was calculated in order not to exceed ten times the acceptable systemic levels (systemic , 10 mg/kg body wt).14 The dosage of cisplatin (20 mg/l extremity volume) (Platinol 0.5 mg/ml, Bristol Myers Squibb, Weesp, The Netherl ands) used in the perfusion had been established in a previous study and was based on a maximum tolerable dose of 30 mg/l extremity volume.15 Cisplatin was added to the circulated perfusate in 10 min. During perfusion, serum TNFaand tot al ci spl ati n l evels were determined in the regional and systemic circulation
at 0, 5, 15, 30, 45, 60, 75 and 90 min by ELISA and flameless atomic absorption spectophotometry (FAAS), respectively. The perfusion time was 1 h, followed by wash-out of the extremity with 3 l of Isodex. Tourniquet, cannulas and clips were then removed and the incisions in the vessels repaired. Protamine hydrochloride (Hoffman La Roche,
Mijdrecht, The Netherlands) was administered, to neutralize heparin, in a ratio of 1:1 to the initial dose
of hepar in. All dogs were closely observed for at least 24 h. No anti-inflammatory or analgesic drugs were
administered during follow-up.
All dogs were followed for local and systemic side effects of TNFa and cisplatin perfusion, as well as survival.

Results

Table 1 shows the characteristics of the seven experiments in six dogs. During the experiments conditions for perfusions (pH, PCO 2 , PO 2 ) were kept within the physiological ranges, as in human perfusions. Figure 1 shows the flow, blood pressure, perfusion pressures, weight gain or loss of the extracorporeal circuit and temperature during 60 min of perfusion in the seven experiments.