Abstract

The Family Bucerotidae (hornbills) is characterized by a unique anatomic structure known as the casque. To date, nine great Indian hornbills from multiple US zoological institutions have presented with fatal, invasive squamous cell carcinoma (SCC) originating in the casque. Multiple treatment protocols have been attempted with limited success including radical surgical resection, radiation, and photodynamic agents.^3,10 In this case, antiangiogenic treatment, OLCAT-005a, was selected to suppress neoplasia growth in addition to radiation treatment. The bird eventually succumbed to complications associated with a prosthesis, but this is the first application of topical antiangiogenic agents in avian SCC.

Introduction

The great Indian hornbill (Buceros bicornis) is at risk for invasive SCC of the casque, which in the case literature has been invariably fatal. Nine specimens have been diagnosed, with no survivors. Of the nine affected individuals, six were male, suggesting a gender predisposition. Limited information on the structural and functional anatomy of the casque is documented, but case reports indicate that SCC of the casque is resistant to conventional treatments used in humans and other mammals.^8,12,14

Angiogenesis (neovascularization) is a critical event in neoplasia formation, allowing for rapid neoplasm expansion and metastases.^1,5 Microvasculature density correlates positively with malignant progression in squamous neoplasia, and cutaneous neoplasms show increased neovascularization.^4,7 Antiangiogenic therapy is a new treatment modality designed to suppress tumor blood vessel growth, halt neoplasia progression, and improve overall survival.

Case Report

In June 2003, a 33-year-old male great Indian hornbill presented for examination because of a malodorous smell. Physical exam revealed weight loss and a soft necrotic area on the cranial casque, containing large amounts of pus and secondary myiasis. Blood was collected for baseline complete blood count, chemistry panel, and Aspergillus titers (Table 1). Lateral and ventrodorsal skull radiographs revealed a large soft tissue density occupying the entire casque, extending to the supraorbital rim of the cranium. Multiple punch biopsies were collected for culture and histopathology (NWZP, Northwest Zoo Path, Monroe, WA 98272, USA) revealed invasive SCC. The wound was aggressively debrided. Proteus but no fungal organisms were cultured from the debrided material. Enrofloxacin (Baytril, Bayer Corporation, Shawnee Mission, KS 66225, USA, 10 mg/kg IM, SID, for 7 days) was initiated.

Table 1. Primary focus of clinical pathology reported every 2 weeks

One week post-presentation, after preliminary histopathology returned with SCC, the bird was transported to LSU to undergo radical casque resection. The bird was anesthetized with sevoflurane (SevoFlo, Abbott Laboratories, Chicago, IL 60064, USA). The dorsal casque was resected in a semi-circle using a bone saw, resulting in exposed spongy bone. The wound was lavaged thoroughly with 0.5% chlorhexidine (Nolvasan, Fort Dodge, Fort Dodge, IA 50501, USA) and bandaged with Tegaderm (3M, Saint Paul, MN 55144, USA). Recovery from anesthesia was uneventful. Enrofloxacin was continued orally twice daily and Benebac (Bird Benebac, Pet Ag Inc. 261 Keyes Ave., Hampshire, IL 60140, USA; 2 g PO, SIS) was initiated. Following this surgery, the bird appeared bright and alert, with a good appetite and weight maintenance.

One week post-surgical resection radiation, using a 6MV linear accelerator, was initiated while the bird was in sedated as before, placed in sternal recumbency, and aligned using orthogonal positioning lasers. Prior to each of the three weekly radiation treatments, the casque was aggressively debrided and cleaned with 0.5% chlorhexidine solution. To allow for an adequate radiation build-up zone, 1 cm of tissue equivalent material was placed over the neoplasm bed. The total field size was 6.1 cm×19.4 cm and included a 3-cm margin. Radiation (10 Gy) was administered through lateral parallel opposed fields, with the prescribed dose delivered to the midline. The post-radiation wound was bandaged with combination of a Tegaderm and Duoderm (Conva Tec Ltd, Bristol Myers Squibb, Princeton, NJ 08543, USA). Twice weekly assessments were performed and, as needed, wound debridement, lavage, and bandage changes were applied. Blood was collected every 14 days to monitor clinical pathology profiles (Table 1). Leukocytosis prompted blood cultures that demonstrated Pseudomonas bacteremia. Systemic antibiotics were resumed (Enrofloxacin, 10 mg/kg, PO, BID for 5 days).

The hornbill was returned to LSU for ablation with a CO₂ laser, to remove the casque base and dorsal rhinothecal sinus before radiation treatment was resumed. After 2 weeks, surgical excision was performed to remove the neoplasm visible on ventrodorsal and lateral skull radiographs. One third of the dorsal rhinotheca was removed by blunt dissection and electrocautery. Histopathology revealed multifocal osteolysis, fibroplasia, and inflammation with small nests of SCC embedded in inflamed and necrotic tissue. Although the animal appeared clinically improved 8 days after surgery, skull radiographs revealed lysis of the supraorbital ridge while visible neoplasm was again apparent on the casque.

At this time, topical antiangiogenic therapy was initiated using the regimen OLCAT (off-label combinatorial antiangiogenic therapy)-005a developed by the Angiogenesis Foundation using FDA-approved drugs that show biologic activity on vascular endothelial cells or neovascularization.⁹ The OLCAT-005a regimen includes imiquimod 5% cream, tretinoin 0.1% microsphere gel, calcipotriene 0.005% ointment, diclofenac 3% gel in hyaluronic acid, and hydrocortisone valerate 0.2% ointment. Drugs were mixed together in equal parts prior to applying a thin layer over the affected tissue. The frequency of administration was governed by the individualized maximal tolerated dose (IMTDSM) algorithm developed by the Angiogenesis Foundation17: 2×/wk×2 wk, 3×/wk×2 wk, Monday through Friday ×2 wk, then daily (total: 12 wk of ≥3×/wk).

Decrease in neoplastic lesion size was visibly apparent after only 1 week of topical antiangiogenic therapy. Two weeks after topical therapy was initiated, the first negative biopsy returned. Radiographs at this time suggested no apparent neoplasia and clinical pathology had returned to normal limits.

Due to the multiple casque resections, the rhinotheca became weakened and multiple procedures were attempted to enhance the structural support. The antiangiogenic treatments were decreased in frequency to minimize excessive stress while the LSU Dental service and a local artisan were consulted for the creation of a prosthetic casque.

One week following cessation of the 12-week topical protocol, visible regrowth of the neoplasm in the left rostrum was noted. As this area had not been treated directly with the topical agents and biopsy for histopathology revealed SCC, topical treatment was re-instituted. With the prosthesis nearing completion, final adjustments included correcting the weight in comparison with the original casque and adding portals that would allow application of topical treatments. The prosthetic composition was a rigid Urethane polymer (GTS-850, Industrial Polymer Inc., Houston, TX 77047, USA).

At 15 weeks post-treatment, the bird was manually restrained for prosthetic placement. At this time, visible re-growth of neoplasm was debrided, the wound lavaged with dilute chlorhexidine, and topical treatment resumed. Skull radiographs revealed lytic and proliferative lesions cranial to the frontal bone. The heated prosthesis was appropriately molded and screwed into the soft palate with a titanium plate. When returned to holding facility, the bird appeared slightly off balance, yet was attempting to catch grapes. Within 36 hours of placement, the bird managed to dislodge the prosthesis. A second prosthetic was attached with a larger titanium plate that was removed by the bird within days.

The bird was assist-fed until transported to LSU after this event for the final surgical resection and prosthetic placement. The remaining affected casque was removed and the third prosthesis was attached using 18” gauge cerclage wire looped through the remaining viable casque. Of note, this prosthetic was heavier than the first two. Three days post-operatively, the bird appeared depressed with cervical ventroflexion observed. The bird was manually restrained to remove part of the prosthesis (350 g). Severe bruising of the hocks and subcutaneous hematomas were visible. The following day, the bird remained depressed in mentation so supportive care and assist feeding were required. The bird expired hours after this treatment with a presumptive diagnosis of exertional myopathy with necropsy revealing severe visceral gout and pectoral zonal myositis. However, no apparent signs of neoplastic growth or metastasis were observed grossly while histopathology of the rhinotheca revealed SCC with no vascular invasion.

Results and Discussion

SCC in the great Indian hornbill is an extremely aggressive disease that does not respond completely or consistently to conventional treatments such as surgical resection, radiation, and photodynamic agents. On presentation, an affected bird may appear as a severe fungal or bacterial casque infection. Skull radiographs and multiple deep punch biopsies are required for diagnosis. It is recommended that all great Indian hornbills have multiple (lateral, ventrodorsal, rostrocaudal) radiographic views of the skull performed once to twice yearly to aid in early detection (www.coraciiformestag.com).

Neoplasia is dependent upon angiogenesis for expansion and metastases. Currently, there are more than 70 antiangiogenic drugs in human oncology clinical trials. In this particular case, a novel treatment was developed (OLCAT-005a) by the Angiogenesis Foundation suppressed malignant growth. Each drug component in the OLCAT-005a protocol has discrete antiangiogenic mechanisms, as well as synergistic biologic effects.⁶ Imiquimod exerts anti-angiogenic activity by up-regulating of local production of interferons and interleukin-12 which have anti-proliferative and pro-apoptotic effects on endothelial cells.^13,16 Retinoids such as tretinoin, vitamin D analogs such as calcipotriene, and COX inhibitors such as diclofenac, have been shown to have antiangiogenic activity.^9,11,15 The combined additive and synergistic actions of these components in OLCAT-005a have biologic and clinical activity in treating SCC.¹⁶

This is the first case of successful application of antiangiogenic therapy to an avian SCC. During the 12-week course of topical treatment, neoplasia suppression was noted in treated areas previously recalcitrant to surgery and irradiation. Additionally, the neoplasm re-appeared in the sites in which the topical agents were not applied or unable to penetrate completely. The neoplasm recurred after cessation of the OLCAT-005a although the final histopathology revealed no signs of vascularization within the recrudescent mass. Therefore, long-term angiostatic treatment may be beneficial to suppress SCC growth to convert this otherwise fatal disease into a manageable condition. The agents appear safe, well tolerated, and demonstrated efficacy for neoplasia suppression in avian SCC. As more data regarding the anatomic structures and functions of the Bucerotid casque are determined, diagnostics and preventive medicine will improve for this structure.

Acknowledgments

The authors thank the dedication of the Audubon Bird and Veterinary Departments.

Literature Cited

1.  Folkman, J. 1995. Angiogenesis in cancer, vascular, rheumatoid, and other diseases. Nat. Med. 1:27–31.

2.  International Species Information System. 12101 Johnny Cake Ridge Road, Apple Valley, MN 55124, USA. www.isis.org

3.  Leach, M.W. 1992. A survey of neoplasia in pet birds. Sem. Av. Exotic Pet. Med. 1:52–64.

4.  Li, V.W., R. A. Ball, W. W. Li, and R. Barnhill. 2003. Microvessel density in actinic keratosis and squamous cell carcinoma and treatment by angiogenesis inhibition. Proc. Am. Acad. Dermatol: 520.

5.  Li, W.W. 2000. Tumor angiogenesis: molecular pathology, therapeutic targeting, and imaging. Acad. Radiol. 17(10):800–811.

6.  Lingen, M.W., P. J. Polverini, and N.P. Bouck. 1998. Retinoic acid and interferon alpha act synergistically as antiangiogenic and antitumor agents against human head and neck squamous cell carcinoma. Cancer Res. 58:5551–5558.

7.  Loggini, B., L. Boldrini, S. Gisfredi, S. Ursino, T. Camacci, K. DeJeso, G. Cervadoro, R. Pingitore, P. Barachini, P. Leocata, and G. Fontanini. 2003. CD34 microvessel density and VEGF expression in basal and squamous cell carcinoma. Pathol. Res. Pract. 199: 705–712.

8.  Manger Cats-Kuenen, C.S. 1961. Casque and bill of Rhinoplax vigil (Forst) in conjunction with the architecture of the skull. Verhandelingen Der Knoninklijke Nederlandse Akademie Van Wet-enschappen, Afd. Natuurkunde, NV Noord-Hollandsche Uitgevers Maatschappij, Amsterdam. 1–47.

9.  Mantell, D.J., P.E. Owens, N. J. Bundred, E. B. Mauer, and A. E. Canfield. 2000. 1 α,25-dihydroxyvitamin D(3) inhibits angiogenesis in vitro and in vivo. Circulation Res. 87:214.

10.  Miller, R.E., D.W. Trampel, W.J. Boever, and M.A. Kling. 1985. Squamous cell carcinoma in a great Indian hornbill (Buceros bicornis). J. Zoo Anim. Med. 16:131–136.

11.  Rivers, J. K., J. Arlette, N. Shear, L. Guenther, W. Carey, and Y. Poulin. 2002. Topical treatment of actinic keratoses with 3.0% diclofenac in 2.5% hyaluronan gel. Br. J. Dermatol. 146:94–100.

12.  Roberts, W.G., M.K. Klein, M. Loomis, S. Weldy, and M.W. Berns. 1991. Photodynamic therapy of spontaneous cancers in felines, canines, and snakes with chloro-aluminum sulfonated phthalacyanine. J. Natl. Cancer Inst. 83:18–22.

13.  Sidbury, R., N. Neuschler, E. Neuschler, P. Sun, W. Xia-qi, R. Miller, M. Tomai, E. Puscasiu, S. Gugneja, and A. S Paller. 2003. Topically applied imiquimod inhibits vascular tumor growth in vivo. J. Invest. Dermatol. 121:1205–1209.

14.  Suedmeyer, W.K., D. McKaw, and S. Turnquist. 2001. Attempted photodynamic therapy of squamous cell carcinoma in the casque of a great hornbill (Buceros bicornis). J. Avian Med. Surg. 15:44–49.

15.  Vega Diaz, B., M. C. Lenior, A. Ladoux, C. Frelin, M. Demarchez, and S. Michel. 2000. Regulation of vascular endothelial growth factor expression in human keratinocytes by retinoids. J. Biol. Chem. 275:642–50.

16.  Zand, S., W. W. Li, and V. W. Li. 2004. Efficacy of topical antiangiogenic therapy for basal cell and squamous cell carcinoma: a case series of 123 lesions. J. Amer. Acad. Dermatol 50: 8.

17.  Zand, S., W. W. Li, and V.W. Li. 2004. IMTD: An algorithm-based approach to optimize dose-response in the treatment of skin cancer using imiquimod. J. Amer. Acad. Dermatol. 50: 129.