Retrospective Study of 33 Dogs Diagnosed with Osteosarcoma (OSA) by Fine Needle Aspiration Cytology (FNAC) at FMVZ Vet Hospital, UNESP- Botucatu, SP

Aim of Study : Evaluate cases of dogs with osteosarcoma, when diagnoses using FNAC. Study Design: A retrospective study, from archive at Veterinary Hospital, selected 33 dogs of different races, 23 females (69.6%) and 10 males (30.3%) were addressed, the age of tested dogs ranged from 1 to 15 years, with diagnoses of osteosarcoma using FNAC. These animals are classified according to localization of tumor, cytopathology evaluation and radiography exam. Results: In this study, a general OSA prevalence on appendicular skeleton of 90.9% (30), being humerus and axial skeleton 9.1% (3). Likewise, the highest age incidence ranged from 6 to 15 years (78.7%). 33 of 24 animals (72.7%) had tumor complications in forelimbs discriminated from high to low incidence: 17 (51.5%) left forelimbs (LFL,) and 7 (27.3%) right forelimbs (RFL). Cancer signs also appeared in axial skeleton of 2 individuals (6.5%), and paranasal sinus of another Original Research Article Wilmsen et al.; ARRB, 9(1): 1-8, 2016; Article no.ARRB.22707 2 (3.2%). Cytoplasmic and nuclear characteristics showed atypical mitosis in 31 (93.9%), and multinucleated cells in 33 (100%) on evaluated specimens. Conclusion: The use of FNAC demonstrated to be a simple, quick, efficient, minimally invasive, and inexpensive diagnostic tool.


INTRODUCTION
Osteosarcoma (OSA) is the most common primary bone tumor in dogs, having a malignant mesenchymal origin. Highly aggressive and invasive, OSA is initially confined to local bone microenvironment, but also involves distant organs according to tumor progression [1,2]. This cancer usually affects long bones with rapid growth, which eventually causes early death due to metastatic potential mainly on lungs [3]. Compromised bone site undergoes rapid resorption, resulting in the release of growth factors and cytokines. Such condition accelerates vascularization and favors metastasis [4].
Thus, cortical lysis and periosteal damage appear due an exacerbated activity and proliferation of osteoblast cells. Consequently, bone pain comes after being compromised its structural integrity [5]. Because similarities in biology and tumor treatment, major differences between dogs and humans are based on the age of occurrence, location, and use of chemoprophylaxis [6].
Three osteosarcoma locations are commonly found in the canine skeletal system: long bones, axial skeleton, and extraskeletal manifestation. In long bones (appendage), 75% of neoplasia cases involve humerus, femur, radius, ulna and tibia [7]. Axial osteosarcoma has 24% of incidence, mainly in the skull bones, spine, and pelvis [8]. Extraskeletal cases represent only 1% of this cancer in dogs [9]. Some breeds (medium, large, and giant) have higher diagnostic rates (Rottweiler, Saint Bernard, Great Dane, Irish Setter, Doberman, German Shepherd, Golden Retriever, and Greyhounds). However, predisposition is related to size, not to race [10,11].
Oncological diagnosis in veterinary medicine still lack effectiveness, which directly jeopardizes survival of cancer patients. Therefore, this review emphasizes the use of Fine Needle Aspiration Cytology (FNAC) as a precise and rapid diagnostic method for osteosarcoma lesions.
This medical tool has become more prevalent in recent years as a less invasive, simple, accurate, and low-cost medical procedure, with high ambulatory applicability and a rapid diagnostic response. In addition, the implied absence of anesthetic procedures benefits critically ill patients. Quality and quickness of examination results increase treatment success by enabling medical staff to adopt suitable and opportune therapeutic measures [12].
FNAC is already widely used for diagnosis of hyperplasia, inflammation, cancer and degenerative diseases. In veterinary medicine, it has been a helpful technique for oncological diagnosis since the 1980s [13].
Nowadays, FNAC is considered as a technological breakthrough to scrutinize bone neoplasm in animals [13,14]. In conjunction with traditional diagnostic methods, it may favor tumor staging when aggregated to the clinical treatment protocol [15]. Other advanced imaging technologies such as scintigraphy, computed tomography and magnetic resonance allow a more detailed assessment of features and extent of the malignancy. Nevertheless, these tests are not always available [16].
According to WYPJI, 2011, FNAC sensitivity ranges from 92 to 97% when applied in bone tumors. In this way, this study aimed to evaluate the use of fine needle aspiration cytology as a primary diagnosis tool to assess osteosarcoma cases attended by the Veterinary Hospital of the Faculty of Veterinary Medicine and Zootechny, FMVZ-UNESP, Botucatu.

MATERIALS AND METHODS
From January 2010 to June 2015, a retrospective study of osteosarcomas cases was carried out in dogs treated at the Veterinary Hospital of the Faculty of Veterinary Medicine and Zootechny, FMVZ-UNESP, Botucatu. 33 dogs selected of different breeds, 23 females and 10 males were addressed, the age of tested dogs ranged from 1 to 15 years. Information was obtained on clinical and radiological examination (Figs. 1, 2), and then sent to the pathology service to collect samples for cytological examination and diagnosis of osteosarcoma (Table 1).
FNAC technique was performed through 10-ml syringes with 25x7 mm disposable needles coupled to a Valery cytoaspirator. Sampling place was submitted to antisepsis, according to hygienic and sterilization protocols. Then, localized punctures were executed by needle insertion, followed by continuous aspirations and a quick "fan" positioning to obtain specimens by capillary action.

RESULTS AND DISCUSSION
OSA diagnostic prevalence of appendicular skeleton in the specimens was 90.9% (30), and axial skeleton 9.1% (3). No case of extraskeletal manifestation was recorded.
The main symptoms reported by animal owners at consultation time were claudication (21), swelling in compromised regions (12) and hyporexia (4). These results corroborate those acute or chronic claudication, and localized edema in the affected limb [19].
Pain was present in all animals during physical examination. However, owners did not frequently identify such a condition. Sometimes animal avoids supporting affected limb due to pain caused by affected area augmentation. Pain and lameness may be related to microfractures, periosteum rupture, and major bone fracture, according tumor evolution [20][21][22][23].
Studied dogs belonged to largest type, OSA frequency in large and giant breed dogs is on average 125 times greater in dogs weighing more than 36 kilograms than those with 9 kilos and less [24][25][26].
The highest frequency of OSA in dogs occurs at forelimbs, which bear 60% of body weight, followed by hindlimbs. In this research, 33 of 24 assessed specimens (72.7%) had tumor at forelimb. Results clearly showed a major incidence (51.5%) on left forelimb (LFL), with 17 cases, than those on right forelimb (RFL), with 7 (27.3%). These results match those found by Silveira et al. [30]: 40% of positive outcomes in LFL, and 34% in RFL [33,34].
Proximal humeral region had the higher proportion of lesions. 16 of the 24 animals showed tumors in this area (66.6%), and 8 (33.3%) at the distal portions of radius/ulna, with joint affection. These findings are consistent with those pointed out in previous investigations, 60% of osteosarcoma cases are located in such bone area, followed by the distal region of femur. In the present study, the latter only occurred in 3 cases (9,07%) [21,25,30,34].
When seeking possible metastatic foci by radiological evaluation, 25 (86.2%) of 29 dogs showed no change, and 8 (27.5%) had nodules highly suggestive of pulmonary metastasis. Identifying lung metastases is possible in less than 10% cases at the consultation time [37]. In summary, FNAC was performed in the attended animals (33) to evaluate cytoplasmic characteristics, nuclear, chromatin condensation, giant cells and atypical mitosis, beyond malignancy criteria (Fig. 3). Atypical mitosis cells were found in 31 specimens (93.9%), and multinucleated cells in all of 33 (100%). Cytology enables to demonstrate neoplastic processes by means of microscopic cell evaluation [33,38].
Regarding nuclear features, clear nucleoli were observed in 29 cases (87.8%) and anisocariose in 30 (90.9%) (Fig. 4) 14 animals (42.4%) underwent surgery (affected limb amputation). The amputation is the main treatment choice to remove the major focus of pain. Due to the advanced stage of cancer, 17 (51.5%) animals were euthanized because poor prognosis. Only one animal required limb amputation surgery and chemotherapy. Limb amputation, combined with an appropriate chemotherapy, as the best therapy to increase animal survival. Likewise, one animal did not return to the clinic for service sequence after primary consultation and diagnosis [33,39].

CONCLUSION
Facing poor survival of OSA patient and limited therapeutic options, the use of FNAC demonstrated to be a simple, quick, efficient, minimally invasive, and inexpensive diagnostic tool. Given its importance for veterinary medicine, the findings suggest that further studies should be done in order to standardize and improve FNAC method.

CONSENT
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ETHICAL APPROVAL
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