Cytologic features of oral squamous cell carcinoma in an indo-pacific bottlenose dolphin (Tursiops aduncus): Papanicolaou stain and immunocytochemistry using liquid-based cytology

211

1. Snow AN, Laudadio J. Human papillomavirus detection in head and neck

212

213

214

squamous cell carcinomas. Adv Anat Pathol. 2010; 17:394-403.

2. Bossart GD, Ghim S, Rehtanz M, et al. Orogenital neoplasia in Atlantic bottlenose

dolphins (Tursiops truncatus). Aquat Mamm. 2005; 31:473-480.

215

3. March DT, Blyde DJ, Bossart GD, et al. Piroxicam and doxycycline treatment for

216

an oral squamous cell carcinoma in an inshore bottlenose dolphin (Tursiops

217

aduncus). Aust Vet J. 2016; 94:203-207.

218

4. Navone R, Burlo P, Pich A, et al. The impact of liquid-based oral cytology on the

219

diagnosis of oral squamous dysplasia and carcinoma. Cytopathology. 2007;

220

18:356-360.

221

5. da Silva AD, Daroit NB, Cardoso FB, et al. Epithelial oral mucosal cells: Do they

222

behave differently when exposed to oral carcinogens? Cytopathology. 2018;

223

29:49-57.

224

225

226

6. Wright RG, Halford JA. Evaluation of thin-layer methods in urine cytology.

Cytopathology. 2001; 12:306-313.

7. Murphy PG, Henderson DT, Adams MD, et al. Isolation of RNA from cell lines and

227

cervical cytology specimens stored in BD SurePath preservative fluid and

228

downstream detection of housekeeping gene and HPV E6 expression using real

12

229

230

time RT-PCR. J Virol Methods. 2009; 156:138-144.

8. Molony P, Werner R, Martin C, et al. The role of tumour morphology in assigning

231

HPV status in oropharyngeal squamous cell carcinoma. Oral Oncol. 2020;

232

105:104670.

233

9. Jörundsson E, Lumsden JH, Jacobs RM. Rapid staining techniques in

234

cytopathology: a review and comparison of modified protocols for hematoxylin

235

and eosin, Papanicolaou and Romanowsky stains. Vet Clin Pathol. 1999;

236

28:100-108.

237

10. Sawa M, Yabuki A, Miyoshi N, Arai K, Yamato O. Rapid-air-dry Papanicolaou

238

stain in canine and feline tumor cytology: a quantitative comparison with the

239

Giemsa stain. J Vet Med Sci. 2012; 74:1133-1138.

240

11. Siebers AG, van der Linden H, Vedder JEM, Bekkers RLM, Melchers WLG,

241

Bulten J. Presence of koilocytosis in low-grade smears of high-risk HPV-positive

242

women is a negative predictor for cervical intraepithelial neoplasia grade 3 or

243

more. Cytopathology. 2018; 29:275-280.

244

12. Chun JW, Lee K, Lee SH, et al. Comparison of liquid-based cytology with

245

conventional smear cytology for EUS-guided FNA of solid pancreatic masses: a

246

prospective randomized noninferiority study. Gastrointest Endosc. 2020;

247

91:837-846.

13

248

13. Rivera C. Essentials of oral cancer. Int J Clin Exp Pathol. 2015; 8:11884-11894.

249

14. Shigeishi H, Sugiyama M. Risk Factors for Oral Human Papillomavirus Infection

250

in Healthy Individuals: A Systematic Review and Meta-Analysis. J Clin Med Res.

251

2016; 8:721-729.

252

15. Rehtanz M, Ghim SJ, Rector A, et al. Isolation and characterization of the first

253

American bottlenose dolphin papillomavirus: Tursiops truncatus papillomavirus

254

type 2. J Gen Virol. 2006; 87:3559-3565.

255

16. Van Bressem MF, Cassonnet P, Rector A, et al. Genital warts in Burmeister's

256

porpoises: characterization of Phocoena spinipinnis papillomavirus type 1

257

(PsPV-1) and evidence for a second, distantly related PsPV. J Gen Virol. 2007;

258

88:1928-1933.

259

260

17. Robles-Sikisaka R, Rivera R, Nollens HH, et al. Evidence of recombination and

positive selection in cetacean papillomaviruses. Virology. 2012; 427:189-197.

261

18. Cortés-Hinojosa G, Subramaniam K, Wellehan JFX Jr, et al. Genomic

262

sequencing of a virus representing a novel type within the species

263

Dyopipapillomavirus 1 in an Indian River Lagoon bottlenose dolphin. Arch Virol.

264

2019; 164:767-774.

265

266

19. Cheah PL, Koh CC, Nazarina AR, Teoh KH, Looi LM. Correlation of p16INK4a

immunoexpression and human papillomavirus (HPV) detected by in-situ

14

267

hybridization in cervical squamous neoplasia. Malays J Pathol. 2016; 38:33-38.

268

20. Cortés-Hinojosa G, Subramaniam K, Wellehan JFX Jr, et al. Genomic

269

sequencing of a virus representing a novel type within the species

270

Dyopipapillomavirus 1 in an Indian River Lagoon bottlenose dolphin. Arch Virol.

271

2019; 164:767-774.

272

273

274

275

276

277

278

279

280

281

282

283

284

285

15

286

Figure legends

287

Figure 1. SurePath manual method kit. A positively charged slide (arrow) and a

288

settling chamber (arrowhead) set in a slide rack (circle).

289

Figure 2. The dysplastic cells with koilocytosis (arrow) appear as a sheet-like

290

structure. Papanicolaou staining.

291

Figure 3. The non-keratinized SCC cells appear as overlapping structures.

292

Papanicolaou staining.

293

Figure 4. The keratinized SCC cells displayed a polygonal morphology. Papanicolaou

294

staining.

295

Figure 5. Occasionally, the keratinized SCC cells showed an onion-like cluster.

296

Papanicolaou staining.

297

Figure 6. The SCC cells showed intense nuclear reactivity to p53 protein.

298

Immunocytochemistry.

16

Table 1. Procedure for creating the SurePath slides

Step Procedure

Fix the sample with CytoRichTM Red Preservative Fluid* (30 min)

Centrifuge the specimens at 3,000 rpm (2 min) and remove the

supernatant

Add distilled water (1.5 mL) to the sediment and resuspend it

Transfer the specimen (0.5 mL) to a settling chamber* (3 times) and

mount it on the positively charged slide* (15 min)

Invert the slide rack* and discard the supernatant

Remove the settling chamber and immediately soak in 95% ethanol (30

min)

*: Becton Dickinson

...