Ultrasonography of Osteosarcoma in Dogs
Sonography Atlas of Dogs

Abstract

The biological behavior of osteosarcoma in dogs is similar to that in humans and the dog has been suggested as a model for the disease in humans . Be- cause occult metastatic disease is common at presenta - tion , systemic therapy is necessary . The dihydro - pyridine , dexniguldipine hydrochloride (B 859-35), is a potent inhibitor of protein - kinase - C ( PKC )- stimu - lated cell proliferation and has shown therapeutic activity in experimentally induced neuroendocrine hamster lung tumors and in a mammary cancer cell line. In human osteosarcoma cell lines , PKC activity can be down - regulated , resulting in increased sensitiv - ity to cisplatin . Since these results supported the involvement of PKC inhibitors in the therapeutic man - agement of osteosarcoma , we performed a prospective , randomized clinical trial using dogs with naturally oc - curring appendicular osteosarcoma to determine the therapeutic potential of dexniguldipine . Dogs received either no drug treatment ( control group , n : 8 ), stan - dard treatment ( e . g ., cisplatin , n = 14 ), or dexniguldipine treatment (n = 14 ) following amputation . Dexnigul - dipine - and cisplatin - treated dogs had a longer median remission duration and survival time than untreated dogs ( P < 0 . 05 ); however , dexniguldipine - treated dogs had a shorter survival time than cisplatin - treated dogs ( P < 0 . 05 ). The results of this study demonstrate that dexniguldipine has significant activity in the inhibition of canine osteosarcoma micrometastases . The identi - fication of a tumor model that may be responsive to this class of antiproliferative agents warrants further clinical investigation to determine the optimum dosage of dexniguldipine and the role it may have in the therapeutic management of canine osteosarcoma .

Key words: Osteosarcoma , Canine , Dexniguldipine , Dihydropyridine

Introduction

The biological behavior of osteosarcoma in dogs is similar to that in humans and the dog has been sugges - ted as a model for the disease in humans ( Withrow et al. 1991 ; Hahn et al. 1994 ). Because occult metastatic dis - ease is common at the time of diagnosis ( Spodnick et al . 1992 ), systemic therapy is necessary . The recommended treatment for canine osteosarcoma is removal of the primary tumor ( e . g ., amputation or a limb - sparing pro - cedure ) followed by adjuvant chemotherapy ( O ' Brien et al. 1993 ). The most effective drugs in dogs are cisplatin ( Platinol , cis - diammine - diehloroplatinum II , Bristol - Myers Squibb Oncology Division , Evansville , Ind .) and doxorubicin hydrochloride ( Adriamycin , Adria Labor - atories , Dublin , Ohio ), given as single agents or in combination ( Thompson and Fugent 1992 ; Mauldin et al. 1988 ).

Calcium / calmodulin - and protein - kinase - C ( PKC )- dependent mitogenic signal transduction pathways are expressed in many types of cancer . Dexniguldipine ( dexniguldipine hydrochloride , Byk Gulden Pharma - ceuticals , Konstanz , Germany ) has been shown to in - hibit calcium / calmodulin - and PKC - dependent growth of human lung cancer cells and a mammary cancer cell line in vitro ( Gietzen et al. 1990 ; Uberall et al. 1991 ; Schuller and Orloff 1994 ). It is hypothesized that inhibition of either pathway would stop tumor growth . Because dexniguldipine may inhibit growth of micrometastases , a clinical trial was performed to evaluate the efficacy of dexniguldipine in the treatment of dogs with naturally occurring appendicular os - teosarcoma following amputation .

Materials and methods

Dogs with appendicular osteosarcoma were evaluated prospectively at the University of Tennessee Veterinary Teaching Hospital . Before treatment , all dogs were clinically staged by survey thoracic radio - graphy ( three views) and abdominal radiography ( two views), bone radiographs of the primary tumor site , and bone scintigraphy ( Hahn et al. 1990 ; Lamb et al. 1990 ; Henderson et al. 1995). Tumor size ( volume ) was determined from preoperative radiographs ( Lamb et al. 1990 ; Henderson et al. 1995 ). To identify toxicity attributable to dexniguldipine or cisplatin treatment , all dogs were required to have an absolute peripheral blood granulocyte count above 4000 /~ tl, a platelet count above 150000/ pl , serum urea nitrogen below 30 mg / dl , creatinine below 1 . 8 mg / dl , and urine specific gravity over 1 .025 g / cm prior to entry in the study .

To determine the significance of dexniguldipine treatment in the management of canine osteosarcoma , dogs free of detectable meta - static disease of synchronous primary tumors had the affected limb amputated and were then randomized to receive either no drug treatment ( control group ), standard treatment using cisplatin (240 mg / m 2 cumulative dose ; 60 mg / m 2 given intravenously every 3 - 4 weeks; Knapp et al. 1988 ; Thompson et al. 1992), or dexnigul - dipine (10 mg / kg , given once orally every day for a minimum of 1 year ; Schuller et al. 1988 ; Schuller personal communication 1991 ). Following initiation of the assigned treatment , owners were required to keep a daily diary recording the occurrence and subjectively assessing the severity of vomiting , diarrhea , anorexia , or fever (e .g ., none , mild , moderate , severe ) observed during the study period ( Hahn and Richardson 1995 ).

All dogs were re - staged 1 , 2 , 3 , 6 , 9 , 12 ,15 , 18 , 21 , and 24 months after amputation and / or initiation of therapy . A complete blood count , serum chemistry , urinalysis , and thoracic radiographs ( three views) were obtained at each visitation . When possible , a bone scintigraphic evaluation was performed at similar intervals . Evalu - ation of bone marrow toxicity attributable to drug therapy was based on the following criteria : mild ( granulocyte count < 4000 / gl and / or platelet count < 150000/ gl ), moderate ( granulocyte count < 2000 / gl and / or platelet count < 80000/ pl ), or severe ( granulocyte count < 1000/ gl and / or platelet count < 40000/ pl ). In the event that the neutrophil count was less than 4000 / gl prior to a subsequent cisplatin therapy , the treatment was delayed 1 week and the neu - trophil count re - evaluated . A necropsy was done on all dogs that underwent euthanasia or died. Remission duration was defined as the time from amputation to detection of metastasis ( es ) by physical examination , radiography , or scintigraphy . Survival time was de - fined as the time from amputation to death . Differences in remission duration and survival time between treatment groups (e .g ., dexnigul - dipine versus controls , dexniguldipine versus cisplatin , cisplatin ver - sus controls ) were determined using the Kaplan Meier method and significance was established at P < 0 .05 using the log - rank test .

Results

A total of 54 dogs with appendicular osteosarcoma were brought to the Veterinary Teaching Hospital dur - ing the study period in an attempt to enter 14 dogs in each of the three treatment groups . Eighteen dogs were excluded because metastasis ( es ) were detectable by physical examination (n = 2 ), survey radiography (n = 5 ), or bone scintigraphy (n = 11 ) at presentation . Fourteen dogs were assigned to both the dexnigul - dipine and cisplatin treatment groups . Only 8 dogs were entered into the control ( no treatment ) group because remission durations and survival times ob - served in these dogs during the study period were far shorter than previously reported times ( Spodnick et al. 1992 ). To deny adjuvant treatment (i.e ., dexniguldipine or cisplatin ) was considered inappropriate care for these client - owned , tumor - bearing pets . Subsequent eli- gible dogs were randomized to either dexniguldipine or cisplatin treatment .

The patient population is described in Table 1 . Of the 14 cisplatin - treated dogs , 10 completed the sched - uled four doses of cisplatin ; the other 4 dogs did not complete the scheduled protocol because they de - veloped metastases . A total of 45 doses of cisplatin were given ( mean 3 .2 doses / dog , range 1 - 4 doses ), treatment delay did not occur in any dog during the planned protocol . Kaplan - Meier estimates of remission dura - tion and survival time were determined ( Fig. 1 ). Dex - niguldipine - and cisplatin - treated dogs had a longer median remission duration and survival time than un - treated dogs ( P < 0 .05 for both analyses ). Remission durations were not significantly different between the dexniguldipine - and cisplatin - treated dogs ( P > 0 .05 ). Although dexniguldipine - and cisplatin - treated dogs had a longer survival time than untreated dogs ( P < 0 .05 for both analyses ), the dexniguldipine - treated dogs had a shorter survival time than cisplatin - treated dogs ( P < 0 .05 ). Multivariate analysis showed no sig - nificant relationship of the remission duration or sur - vival time results to age , sex , body weight , tumor stage (i.e ., size ), or cumulative dose of cisplatin .

All dogs underwent euthanasia because of meta - stases . Sites of metastasis were the lungs (n = 33), skel - eton (n = 5 ), and spleen , liver and skin (n = 2 each ). Dogs treated with dexniguldipine had far fewer pul - monary metastases (e .g ., fewer than 10 nodules 0 .5 cm or greater in diameter ) observable at necropsy com - pared to those dogs , given no further treatment or treated with cisplatin , that had multiple pulmonary metastases (e .g ., more than 50 nodules 0 .5 cm or greater in diameter ) at necropsy . However , differences in the number of pulmonary metastatic nodules between the two treatment groups were not significant ( P > 0 .05 ). Bone metastases were documented by radiography (n = 4 ), bone scintigraphy (n = 5 ), and postmortem ex - amination (n = 5 ). Sites of bone metastases were the long bones (n = 2 ), spine (n = 2 ), and ribs (n = 1 ). Four dogs subjected to euthanasia because of bone meta - stases had received dexniguldipine treatment ; the other dog had received cisplatin . Median survival times for dogs developing bone metastases and lung metastases were 28 .6 weeks and 18.1 weeks respectively ; this differ - ence was not significant ( P > 0 .05 ).

Of the 14 dogs treated with cisplatin chemotherapy , 12 developed toxicosis . Toxicoses included vomiting ( n = 12 dogs ) and lethargy (n = 8 dogs ) following one or more cisplatin treatments . Hematological and serum biochemical changes included mild neutropenia ( n = 8 dogs ) and a mild to moderate ( < 60 mg / dl ) increase in blood urea nitrogen concentration (n = 4 dogs ). Al - though , at dosages above 15 mg / kg , dexniguldipine has been shown to cause severe weight loss , vomiting , and / or diarrhea in normal dogs , the 14 tumor - bearing dogs treated with dexniguldipine therapy at 10 mg / kg in this study had no toxicoses .

Fig. 1: A Kaplan - Meier estimates of remission duration for dogs with appendicular osteosarcoma stratified by treatment group. B Kaplan - Meier estimates of survival time for dogs with appendicu- lar osteosarcoma stratified by treatment group. Differences between any two distribution curves ( e . g ., dexniguldipine-treated versus no treatment dogs , cisplatin-treated versus dexniguldipine-treated dogs, etc .) were determined by the log- rank test

Discussion

Dexniguldipine , as used in this prospective , random - ized clinical trial , has antitumor activity in dogs with osteosarcoma .

Because of clinical , radiographic , and pathological similarities , the dog is commonly used as a large - ani - mal therapeutic model for humans with osteosarcoma ( Brodey 1979 ; Misdorp and Hard 1979 ; Hahn et al . 1990 ; Thompson and Fugent 1992 ; Malawer et al . 1993 ; Hahn et al . 1994). Osteosarcoma is estimated to occur in more than 6000 dogs in the United States per year , and that figure may be increasing with the growing popularity of large and giant breeds ( Withrow et al. 1991 ). Dogs with osteosarcoma comprise approxi - mately 1 % - 3 % of all canine malignancies and 85 % - 95 % of all primary bone tumors ( Spodnick et al . 1992 ). Male dogs are affected twice as frequently as female dogs and tumor development occurs with a peak incidence between the ages of 3 and 7 years , although all age groups are affected ( Brodey 1979 ; Misdorp and Hart 1979 ).

In the dog , osteosarcoma commonly occurs in the metaphyses of the long bones ( distal portions of the radius and ulna , proximal portion of humerus , proxi - mal and distal portions to femur and tibia ) and less frequently in the axial skeleton . Forelimb lesions are more common than hindlimb lesions (1.7:1.0). As in humans , common survey - radiographic abnormalities suggestive of osteosarcoma include pronounced corti - cal bone lysis, active , spiculated periosteal new bone formation , and pathological fracture . Definitive diag - nosis of primary , multicentric , metachronous or meta - static osteosarcoma is made by examination of biopsy specimens ( incisional or excisional ).

                                                          Microscopic occult metastatic disease is present in 80 % - 90 % of canine patients at presentation ( Brodey 1979 ; Spodnick et al. 1992 ). In this study , 18 of 54 dogs ( 33 %) presented with macroscopic metastatic disease and in 11 of those 18 dogs ( 61 %) the metastases were occult ( i . e ., not apparent on physical examination and no related clinical signs ) and detected by scintigraphy . Because of the aggressive biological behavior of canine osteosarcoma , some form of systemic therapy is neces - sary if survival is to be improved . Cisplatin has shown exceptional efficacy as an adjuvant to surgery in humans and dogs with appendicular osteosarcoma (Jaffe et al. 1989 ; O ' Brien et al. 1993 ). With amputation alone , the 1 - and 2 - year survival rates are estimated to be 11 . 5 % and 2 % respectively , and the mean and median survival times are estimated to be 19 . 8 and 19 . 2 weeks respectively ( O ' Brien et al. 1993 ). In humans , the 5 - year survival rates following amputation or resection alone are 5 % - 20 % ( Mankin et al. 1991 ). Multiple studies have shown that cisplatin as a single agent markedly improves survival rates in dogs as compared with surgery alone ( O ' Brien et al. 1993 ). With adjuvant cisplatin chemotherapy , 38 % - 62 % 1- year survival rates have been reported . Similarly , the median re- mission duration and survival time for cisplatin - treated dogs in our study was 20.8 weeks and 35.5 weeks respectively , with a i - year survival rate of 35 . 7 %. In humans , the use of multidrug adjuvant and neoad - juvant chemotherapy protocols remarkably improves the 5 - year survival rates to 80 % - 90 % or more ( Mankin et al. 1991 ).

                                                              In this study , the median remission durations and survival times for each of the treatment groups are shorter than the intervals previously reported by others ( Mauldin et al. 1988 ; Thompson and Fugent 1992 ; Spodnick et al. 1992 ; O ' Brien et al. 1993 ). A likely explanation can be attributed to the study design ' s strict adherence of patients to frequent ( e . g ., monthly during the first 3 months and every 3 months there - after ) and rigorous ( e . g ., thoracic and bone radio - graphy , bone scintigraphy ) re - evaluation schedules during the post - amputation period and the use of conservative definitions for remission duration and sur - vival time . Previous studies have not used similar criteria and thus opportunities to detect metastatic lesions prior to the onset of clinical signs or determine the exact time or cause of death are frequently missed . Although the criteria established in this study resulted in the reporting of shorter responses to treatment , the study provided the opportunity for a fair and accurate statistical evaluation of the results to determine the true potential of dexniguldipine in the management of ca- nine osteosarcoma .

Dexniguldipine is a potent inhibitor of PKC - stimu - lated cell proliferation in lung cancer cell lines while it has no effect on cell proliferation in response to activa - tion of cyclic AMP ( Shuller and Orloff 1994 ). Phorbol esters activate PKC by binding directly to the kinase ( Weinstein 1983) while , under physiological conditions , PKC is activated as a second messenger in response to the binding of certain growth factors to their cellular receptors . In human osteosarcoma cell lines , PKC ac- tivity can be down - regulated following continuous ex- posure to the phorbol ester 12 - O - tetradecanoylphorbol 13 - acetate resulting in increased cisplatin cytotoxicity ( Perego et al . 1993 ). These results support the involve - ment of PKC inhibitors , such as dexniguldipine , in the therapeutic management of osteosarcoma .

A dexniguldipine dose of 10 mg / kg was selected for use in this study because results from a phase I dose - escalation study in normal dogs demonstrated that a dose of more than 15 mg / kg , given once orally per day for 4 weeks , resulted in severe weight loss ( 20 % - 25 % reduction in initial body weight ), vomiting , and diarrhea ( Schuller , personal communication ). Be- cause remission durations were not significantly differ - ent between the dexniguldipine - and cisplatin - treated dogs but greater than the remission duration for un - treated control dogs , it is possible that the dosage of dexniguldipine used in this study was not the optimal dose to inhibit the growth of all micrometastases . How - ever , the identification of a naturally occuring large - animal model that may be responsive to this class of antiproliferative agents warrants further clinical invest - igation to determine the optimum dosage of dexnigul - dipine and the role it may have in the therapeutic management of canine osteosarcoma .

Acknowledgements

This work was supported, in part, by a grant from Byk Gulden Pharmaceuticals , D-7750 Konstanz , Germany. Cisplatin was kindly provided by Bristol-Myers Squibb Oncology Division , Evansville , Ind.

The authors would like to acknowledge the assistance of Dr . Karl H. Sanders, Dr . Gregory B . Daniel , Dr . Lina Bravo, Ms . Karen Young, Ms . Regina Barbaro , Mr. James Avenell, and Ms . Marie L . Nolan .

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