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10.5005/jp-journals-10015-1525Koilocytes in Oral REVIEW ARTICLE

Koilocytes in Oral Pathologies 1Preeti Singh, 2Samudrala V Sowmya, 3Roopa S Rao, 4Dominic Augustine, 5Vanishree C Haragannavar, 6Shwetha Nambiar

ABSTRACT How to cite this article: Singh P, Sowmya SV, Rao RS, Augustine D, Haragannavar VC, Nambiar S. in Oral Introduction: Oral HPV affect 1 to 50% of the Pathologies. World J Dent 2018;9(2):149-153. general population. Naturally, about 90% of HPV is eliminated by the immune system, but the ones that persist may result in Source of support: Nil serious . Human papillomavirus is the main cause of Conflict of interest: None at various body sites, such as cervix, uterus, orophar- ynx, head and neck. The prevalence of oral HPV infections in India ranges from 15 to 16%. About 80% of HPV infections are INTRODUCTION present with koilocytosis as an important morphological marker. Human papillomavirus belongs to the family of , Aim: This review focuses on the importance of koilocytes and the Papovaviridae.1 These are small, epitheliotropic its early detection to alert malignant risk for facilitating human double-stranded DNA viruses with more than 120 papillomavirus (HPV)-targeted therapeutic strategies. identified genotypes in human.1,2 There are specific Results: Research in the past has primarily focused on HPV types responsible for the causation of cancerous , as >99% of them harbor HPV. It has been and non-cancerous tumors.3,4 Human papillomavirus observed that the incidence of HPV-associated may be minimized by effective preventive and targeted therapeutic prevalence ranges from 22 to 60% in the normal mucosa modalities. Although oral HPV infections have been minimally that includes subclinical and/or latent infections of the researched lesions such as verruca vulgaris, squamous papil- oral cavity.2-4 loma, focal epithelial and condyloma acuminatum Oral cancers are found to be commonly associated are significantly prevalent in India. The characteristic with HPV with an overall frequency of 74%, observed in all these HPV-associated lesions is koilocytes while 41% showed multiple HPV infections. About 10% as demonstrated using (IHC) with p16 antibody. Although advanced techniques, such as polymerase of men in the age range of 14 to 69 years have an oral HPV 3,4 chain reaction (PCR), deoxyribonucleic acid (DNA) breakage infection compared with 3.6% of women. detection fluorescence in situ hybridization (DBD-FISH), and A higher percentage (more than 40%) is seen in HPV comet assay may confirm their presence, they are cumbersome cancers at the base of tongue and of palatine tonsils.3,5 and not economical. Recent studies have demonstrated that the association Conclusion: Detection of malignancy-prone oral HPV infec- between HPV and OSCC is stronger in young males than tions by the demonstration of koilocytes showing positivity to in females. There is a wide geographical variation in the p16 IHC stain has shown promising avenues in the field of prevalence of HPV among Indians with 27% of oral cancer research for the prediction of diagnosis and prognosis of oral from North India being positive for HPV 16, whereas 25 squamous cell carcinomas (OSCC). to 31% positivity in western India.1,2,6 Clinical significance: Koilocytic cells are pathognomonic of HPV infection. Identification of koilocytes in histopathologic KOILOCYTES sections alerts the pathologist to suspect any underlying HPV infection and directs the surgeon for appropriate treatment. These are squamous epithelial cells that have endured Human papillomavirus infection affects the function or host a number of structural changes due to HPV infection. gene expression that is marked by cellular changes, and The infected cells have an eccentric, hyperchromatic, unmasking of such changes has an important role in the iden- tification and follow-up of such patients. and enlarged nucleus that is displaced by a large perinuclear vacuole showing irregular contour and Keywords: Human papillomavirus, Human papillomavirus- appearing crenated, “raisin-like” or “spoon-like” in associated oral lesions, Koilocytes. shape.7, 8 Hence, the name “” is derived from Greek, in which koilos means “hollow” and “cyte” means cell (Fig. 1).5, 7, 8 1-6Department of Oral and Microbiology, Faculty of Dental Sciences, M.S. Ramaiah University of Applied Sciences The role of cytoplasmic vacuolization in viral rep- Bengaluru, Karnataka, India lication is unclear. However, it could contribute to the Corresponding Author: Preeti Singh, Department of Oral fragility of keratinocytes and release of viral-laden nuclei Pathology and Microbiology, Faculty of Dental Sciences, M.S. from HPV lesions.1,5,8 There is a movement of - Ramaiah University of Applied Sciences, Bengaluru, Karnataka to the superficial layers of the epithelium that is India, e-mail: [email protected] facilitated by disturbing keratin integrity and assembly World Journal of Dentistry, March-April 2018;9(2):149-153 149 Preeti Singh et al

Pathogenesis The actual mechanism by which HPV enters the cell is still unclear. It is accepted that the entry of the virus into the epithelial cell and delivery of DNA into the nucleus is through HPV capsid proteins. Infec- tion occurs when the virus penetrates micro injuries and enters the new host. There are three factors that control the development of incubation phase into active expression: Cell permeability, virus type, and the host immune status.5,9 The sequential steps involved in the of HPV infection have been depicted in Flow Chart 1.

human papillomavirus TYPES Fig. 1: Koilocytes in suprabasal layers of epithelium Currently, around 100 types of HPVs have been identified, but only a few (e.g., HPV 16, 18, 31, and 33) are known to of cornified layer. Collectively these changes are called be associated with malignant risk. Human papillomavi- . Some cells may display binucleation rus types have been broadly categorized into high- and with nuclear atypia and are termed as “koilocytic dys- low-risk types3,4,6 as depicted in Table 1. plasia”.5,7-9 Although koilocytosis denotes an important mor- IMPORTANCE OF KOILOCYTES phological marker for HPV infection, there are instances Koilocytosis is the most common cytomorphologic wherein artifacts caused due to fixation, processing, or change and is considered by pathologists to be a major surgical errors mimic koilocyte-like appearance. histopathologic feature for the determination of HPV

Flow Chart 1: Pathogenesis of HPV infection

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Koilocytes in Oral Pathologies

Table 1: Human papillomavirus types and their significance 13 or 32 with a site specificity of keratinized and nonke- 14 Relative risk Positive HPV types Significance ratinized surfaces respectively, in the superficial layers. High risk Most common: 16 and 18 Oral condyloma acuminatum that commonly affects Others: 31, 33, 35, 39, 45, Associated with buccal mucosa, floor of mouth, lingual frenum, and hard 51, 52, 56, 58, 59, 68 malignancy palate is induced by HPV types 6 and 11. Mild to moderate Low risk 6, 11, 40, 42, 43, 44, 53, 54, Benign 15 61, 72, 73, 81 numbers of koilocytes are appreciated in superficial layer. Oral lichen planus is a chronic immune-mediated , presenting with bilaterally symmetrical lesions infection. In cervical neoplasias, koilocytosis has been that affect the gingiva, lip mucosa, and dorsum of tongue. 9-11 classified as (, 2001): The HPV types 11 and 16 are commonly associated Low squamous intraepithelial lesion • : mild koilocytosis with the lesion with koilocytes predominantly found in with mild /cervical intraepithelial neoplasia stratum granulosum.13 Oral is a precancerous (CIN1). lesion with a malignant transformation rate of 16 to 62%. High squamous intraepithelial lesion • : marked koilocyto- The lesion is most commonly seen on the buccal mucosa, sis with moderate and severe , carcinoma tongue, and palate and may be caused by HPV types 6, in situ /CIN 2 and 3. 11, and 16 and may rarely display koilocytes.2,16 Oral koilocytic dysplasia is a distinct entity that shows Verrucous carcinoma, a variant of squamous cell 8 HPV in light microscopy with 80% accuracy. However, carcinoma, occurring on lips, oropharynx, and larynx, is the clinical connotation and probability for malignant usually associated with HPV 6, 11, 16, and 18. The pres- risk needs to be investigated further. Positive cases of ence of koilocytes is seen in the intermediate layer of the koilocytic dysplasia contain HPV types 31, 33, 51, 16, epithelium.5,6 Oral is usually 2,11 and 18. In HPV infection, koilocytes are a significant associated with HPV 16, 11, and 27 with a frequency of 21 microscopic finding in association with features of con- to 60%. Allocation of koilocytes is usually in the form of 11 ventional . scattered foci throughout the epithelium which suggests A study undertaken by Chaturvedi et al in the United nonclonal origin of tumor nature.6 States evaluated changes in patient characteristics, inci- dence and survival in OSCC sites between HPV-related DEMONSTRATION OF KOILOCYTES IN and HPV-unrelated areas. They concluded that the preva- RELATION TO A SUSPECTED CASE OF lence of HPV-related OSCCs was identified at an earlier VERRUCOUS CARCINOMA USING IHC age group compared with HPV-unrelated OSCCs. Similar results were observed with 2 years survival following Routine and Special Stains 12 radiation therapy. A clinically suspected case of carcinoma that histo- pathologically revealed numerous koilocytic cells in the human papillomavirus-ASSOCIATED superficial epithelium was employed for staining with ORAL LESIONS IHC (p16). Hematoxylin and eosin (H&E)-stained section The prevalence of oral HPV infections in India ranges showed numerous cells in the stratum spinosum and from 15 to 16%.2,13 Koilocytes are generally found in granulosum layers that had a perinuclear halo- and cres- association with HPV oral lesions that include verruca cent-shaped nucleus, suggestive of koilocytes (Fig. 2A). vulgaris, squamous papilloma, focal epithelial hyperpla- Immunohistochemistry using p16 antibody was per- sia, and condyloma acuminatum. formed to confirm the presence of high-risk HPV infec- Squamous papilloma, a benign of squamous tion. Immunohistochemistry showed brown-colored cells, affects predominantly the tongue, buccal mucosa, staining in the cytoplasm, nucleus, and nuclear mem- and lips. Histopathologically, the intermediate layers of brane suggestive of strong positivity for p16 as shown in epithelium demonstrate koilocytes which predominantly Fig. 2B. As the high-risk HPV marker was strongly posi- show positivity for HPV 6 and HPV 7.1,2,13 Verruca vul- tive, the present case was suspected for malignant risk. garis is a common benign skin lesion but can be present Few of the studies favoring our results are as follows: in oral cavity also. The preponderance of mucosal Patil et al17 evaluated the expression of HPV in OSCC and is seen in children and associated with HPV 2 and correlated its association with histological grades using 4 types. The variants of warts could be flat warts associ- p16. The researchers found strong association between ated with HPV types 3 and 10 and butcher’s warts with HPV infection and OSCC. The diffuse pattern of p16 HPV 7. Numerous koilocytes (89%) are often seen in the positivity noted in poorly differentiated OSCC cases superficial spinous layer.13 Focal epithelial hyperplasia/ depicted the increased viral overload, which might have Heck’s disease is a rare benign oral lesion caused by HPV an influence on its aggressive behavior. World Journal of Dentistry, March-April 2018;9(2):149-153 151 Preeti Singh et al

A B Figs 2A and B: Photomicrographs showing (A) koilocytes with crescent-shaped nuclei and perinuclear halo under H&E staining (B) p16 IHC marker showing strongly positive cytoplasmic staining

An IHC evaluation of HPV 16 was done by Agrawal An advanced technique that permits cell-to-cell et al18 using HPV 16 in oral epithelial dysplasias (OEDs) detection and DNA breakage quantification is DBD-FISH and OSCC to ascertain whether the presence of HPV dis- that utilizes whole genome or specific DNA areas. The rupts the regulation of p16INK4A suppressor protein. It purpose of this method is to recognize HPV infection was found that 22.5 and 35% cases were HPV positive in through changes in chromatin due to increase in the OED and OSCC respectively. They concluded that HPV 16 number of alkaline labile sites. These findings may be plays a role in disruption of p16NK4A suppressor protein crucial for the investigation of mechanisms involved in regulation and can be used as a surrogate biomarker for viral tumorigenesis in humans and have been justified identification of HPV infection in OED and OSCC.19 by the study done by Cortés-Gutiérrez et al.21 However, the study by Kim et al20 found contrasting Comet assay or single cell gel electrophoresis is a results in urothelial carcinoma (UC) of the bladder and rapid and highly sensitive fluorescent microscopy–based concluded that the expression of p16 to be a weak sur- method to examine DNA damage and repair at the level rogate marker for the presence of HPV infection in UC of individual cells. This technique is usually applied for of the bladder with squamous differentiation. the detection of protein depletion of HPV. A study by 22 The diagnosis of HPV may be suspected through Cortés-Gutiérrez et al was performed to determine that is characterized by major criteria that the relation of DNA single (ssb) and double (dsb) strand represent koilocytes and minor criteria involving dysker- breaks in women with and without cervical neoplasia atocytes, atypical immature , and binucleation. using comet assays. A significant increase in global DNA As reliable serological tests are not available and damage (ssb + dsb) and dsb was observed in patients culture of the virus is challenging, molecular methods with high-grade squamous intraepithelial lesion (SIL; are the diagnostic tools of choice for validation. 48.90 ± 12.87 and 23.50 ± 13.91 respectively), patients with low-grade SIL (33.60 ± 14.96 and 11.20 ± 5.71 respectively), ADVANCED DIAGNOSTIC TECHNIQUES and controls (21.70 ± 11.87 and 5.30 ± 5.38 respectively), and they concluded that real genomic instability favors Polymerase chain reaction is a molecular technique for women with cervical neoplasia, having higher propor- primer-directed enzymatic amplification of specific target tion of DNA dsb. DNA sequences. Being a noninvasive technique, it is pre- Nucleic acid hybridization technique involves South- 4 ferred to detect the types of HPV. Bouda et al examined ern blot, DB, and reverse dot hybridization methods. It HPV infection in normal oral mucosa, oral , uses radiolabeled nucleic acid hybridization assays to dysplasias, and squamous cell carcinomas using in situ detect HPV infection. Sensitivity of these techniques hybridization, dot blotting (DB) nested PCR, restriction is relatively low compared with PCR. Hence, currently, fragment length polymorphism analysis, and type-spe- these techniques are rarely used.23 cific PCR (TS-PCR). They found positive HPV high-risk Hybrid capture (HC) technology ascertains nucleic types (16, 18) in 98% cases, whereas 2% of OSCC showed acid targets directly using signal amplification to provide positivity for low-risk type (6) of HPV, suggestive of early sensitivity comparable to target amplification methods involvement of high-risk types in oral carcinogenesis. (PCR). It is helpful in the identification of high-risk HPV 152 WJD

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