ALICIA RUMAYOR PIÑA
IDENTIFICAÇÃO DE VÍRUS PAPILOMA HUMANO EM CARCINOMA DE AMÍGDALA E LESÕES EPITELIAIS BENIGNAS DE BOCA, E VÍRUS
EPSTEIN-BARR EM CARCINOMA NASOFARINGEO
IDENTIFICATION OF HUMAN PAPILLOMAVIRUS IN TONSIL CARCINOMA AND BENIGN ORAL EPITHELIAL LESIONS, AND EPSTEIN-BARR VIRUS IN
NASOPHARYNGEAL CARCINOMA
PIRACICABA 2016
IDENTIFICAÇÃO DE VÍRUS PAPILOMA HUMANO EM CARCINOMA DE AMÍGDALA E LESÕES EPITELIAIS BENIGNAS DE BOCA, E VÍRUS
EPSTEIN-BARR EM CARCINOMA NASOFARINGEO
IDENTIFICATION OF HUMAN PAPILLOMAVIRUS IN TONSIL CARCINOMA AND BENIGN ORAL EPITHELIAL LESIONS, AND EPSTEIN-BARR VIRUS IN
NASOPHARYNGEAL CARCINOMA
Tese apresentada à Faculdade de Odontologia de Piracicaba da Universidade Estadual de Campinas como parte dos requisitos exigidos para a obtenção do título de Doutora em Estomatopatologia, na Área de Patologia Thesis presented to the Piracicaba Dental School of the University of Campinas in partial fulfillment of the requirements for the degree of Doctor in Stomatopathology, in Pathology area.
Orientador: Profr. Dr. Oslei Paes de Almeida ESTE EXEMPLAR CORRESPONDE Á VERSÃO FINAL DA TESE DEFENDIDA PELA ALUNA ALICIA RUMAYOR PIÑA, E ORIENTADA PELO PROF. DR. OSLEI PAES DE ALMEIDA
PIRACICABA 2016
O Vírus Papiloma Humano (HPV) e o Vírus Epstein-Barr (EBV) estão associados a lesões epiteliais benignas de boca como papiloma escamoso, verruga vulgar, condiloma acuminado, hiperplasia epitelial multifocal oral e leucoplasia pilosa oral, respectivamente, bem como a um subgrupo de carcinomas de cabeça e pescoço que inclui carcinomas de orofaringe e nasofaringe. A utilização dos termos das lesões epiteliais hiperplásicas benignas de boca tem sido confusa na literatura bem como a relação com o HPV. Em relação aos carcinomas, relatos acerca da prevalência e incidência do HPV e EBV em países como Guatemala e Brasil são escassos. O objetivo deste trabalho foi identificar por hibridização in situ a presença de HPV em carcinoma de orofaringe e lesões epiteliais hiperplásicas benignas de boca, e de EBV em carcinoma de nasofaringe em pacientes oriundos de diferentes serviços de patologia oral e de cabeça e pescoço da Guatemala e Brasil. Para identificar o HPV foram utilizadas sondas de amplo espectro e específicas para os subtipos 6/11 e 16/18, enquanto que para EBV foi utilizada a sonda EBER que detecta os transcritos EBER1 e EBER2. Além disso, foi realizado estudo imunoistoquímico para os anticorpos Ki67, LMP-1 (EBV) e p16 em casos selecionados. Os resultados mostraram prevalência dos subtipos HPV 6/11 em lesões epiteliais hiperplásicas benignas de boca e dos subtipos HPV 16/18 nos carcinomas de amígdala. Todos os carcinomas de nasofaringe do subtipo não queratinizante indiferenciado mostraram positividade para EBV, independentemente da origem geográfica. Tanto na amígdala quanto na nasofaringe, a origem dos carcinomas parece ser no epitélio das criptas, também chamado de epitélio reticular. Interessante que a principal característica destes tumores é a morfologia não queratinizante, e em alguns casos principalmente de nasofaringe, a presença de abundantes linfócitos entre as células neoplásicas é muito semelhante ao que acontece no epitélio reticular normal. Nossos achados indicam que em carcinomas de amígdala e nasofaringe da Guatemala, a incidência tanto do HPV quanto do EBV, respectivamente, foi maior quando comparada aos casos do Brasil, entretanto as razões para estas diferenças ainda precisam ser melhor determinadas.
Palavras-chave: Vírus Papiloma Humano. Vírus Epstein-Barr. Amígdala. Nasofaringe. Hibridização in situ.
Human Papilloma Virus (HPV) and Epstein-Barr Virus (EBV) are associated to oral benign epithelial lesions as squamous papilloma, verruca vulgaris, condyloma acuminatum, multifocal epithelial hyperplasia and hairy leukoplakia, respectively, as well as a subset of head and neck carcinomas that include tonsillar and nasopharyngeal carcinomas. In literature, the terminology used for the oral epithelial benign hyperplastic lesions is confuse, as well as the relationship with HPV. Regarding to carcinomas, reports on the prevalence and incidence of HPV and EBV in countries such as Guatemala and Brazil are scarce. The aim of this study was to identify by in situ hybridization the presence of HPV in oropharyngeal carcinoma and hyperplastic epithelial benign oral lesions, and EBV in nasopharyngeal carcinoma in patients from oral and head and neck pathology services in Guatemala and Brazil. HPV identification was made using a broad-spectrum and specific probes for subtypes 6/11 and 16/18, while EBV was identified using the EBER probe, that detects the EBER1 and EBER2 transcripts. In addition, in selected cases, immunohistochemistry was performed using Ki67, LMP1 and p16. The results showed prevalence of HPV 6/11 in epithelial benign hyperplastic oral lesions and HPV 16/18 in tonsillar carcinomas. All the undifferentiated nonkeratinizing nasopharyngeal carcinomas showed EBV positivity regardless of geographic origin. In both, tonsil and nasopharynx, the origin of these carcinomas seems to be the crypt epithelium, also called reticular epithelium. Interestingly, the main characteristic of these tumors is the nonkeratinizing morphology, and in some cases, mainly from nasopharynx the abundant presence of lymphocytes among the tumor cells, which is very similar to what happens in the normal reticular epithelium. Our findings indicate that in tonsillar and nasopharyngeal carcinomas from Guatemala, the incidence of both, HPV as EBV, respectively, was higher than in those from Brazil, however, the reason for these differences still need to be better determined.
Key Words: Human papillomavirus. Epstein-Barr Virus. Tonsil. Nasopharynx. In situ hybridization.
2 ARTIGOS 16
2.1 Artigo: Human papillomavirus in tonsillar squamous cell carcinomas from Guatemala and Brazil 16
2.2 Artigo: Benign epithelial oral lesions related to human papillomavirus, overlaps and difficulties in diagnosis 30
2.3 Artigo: Epstein-Barr Virus in nasopharyngeal carcinoma of Guatemalan and Brazilian patients 49
3 DISCUSSÃO 60
4 CONCLUSÃO 64
REFERÊNCIAS 65
ANEXOS Anexo 1 – Certificado do Comitê de Ética em Pesquisa 72
Anexo 2 – Declaração da editora 73
1 INTRODUÇÃO
1.1 VÍRUS PAPILOMA HUMANO E CARCINOMA ESCAMOCELULAR DE AMÍGDALA
O carcinoma escamocelular (CEC) de orofaringe associado ao vírus papiloma humano (HPV) é reconhecido por apresentar características clínicas, microscópicas e moleculares particulares, além de ter sido relacionado com um melhor prognóstico. Informação específica de incidência e prevalência deste subgrupo de tumores de cabeça e pescoço não está bem documentada, já que usualmente são agrupados com o carcinoma oral, muito menos ao falar exclusivamente da tonsila, já que eles são agrupados geralmente com outras regiões da orofaringe. Estes tumores são originados principalmente na amígdala palatina de homens, histologicamente são caracterizados por uma morfologia indiferenciada, não queratinizante. Diferente do carcinoma oral convencional, o qual afeta comumente pacientes fumantes com histórico de consumo de álcool, e que histologicamente apresenta uma morfologia queratinizante e não é relacionado com HPV (El Mofty & Lu, 2003; Adelstein et al., 2009). A anatomia da amígdala palatina com cerca de 10 a 30 criptas revestidas por um epitélio distinto ao de superfície, chamado de epitélio reticular, pode ter relação com a maior susceptibilidade à infecção pelo HPV. Tem sido discutido que o carcinoma de amígdala associado à HPV seja originado a partir desse epitélio reticular e que aqueles carcinomas HPV negativos sejam originados do epitélio de superfície. De fato, a similaridade histológica do epitélio reticular e o carcinoma associado à HPV é interessante. O epitélio reticular encontra-se revestindo as criptas e é caracterizado por ser não queratinizante e ter uma aparência basalóide, características encontradas no carcinoma de amígdala associado à HPV (Syrjänen, 2004; Begum et al., 2005; Westra, 2012; Lewis & Chernock, 2014). Portanto, é importante na classificação morfológica inicial definir a provável origem histológica do tumor amigdaliano, se é da cripta ou se é extensão de um carcinoma originado do epitélio da superfície amigdaliana ou de áreas adjacentes como o pilar amigdaliano (Kim et al., 2007). Nos casos de carcinoma não queratinizante metastático a linfonodos cervicais a identificação do HPV é útil na localização do tumor primário, por exemplo, lesões com morfologia indiferenciada, tipo linfoepitelial, indistinguível da morfologia do carcinoma nasofaríngeo, a negatividade para o vírus Epstein-Barr (EBV) e a expressão do HPV indicam origem amigdaliano, enquanto que a expressão do primeiro é indicativa de
origem em nasofaringe (Singhi et al., 2010; Singhi & Westra, 2010; Carpenter et al., 2011; Yasui et al., 2014; Fotopoulos & Pavlidis, 2015).
Na literatura mundial, a associação entre o HPV e carcinoma de amígdala parece ser muito variável entre diferentes regiões, mas no geral a maior parte dos estudos reportam que entre 40% a 65% dos casos são relacionados ao HPV, com o subtipo 16 sendo consistentemente o mais prevalente. Os subtipos 16 e 18 são conhecidos já pela associação com carcinoma de colo uterino, pênis, vulva, vagina e anus. Não existem dados disponíveis de incidência de carcinoma de orofaringe associado ao HPV na Guatemala (Syrjänen, 2004; Kreimer et al., 2005; Rusan & Ovesen, 2012; Forman et al., 2012; de Martel et al., 2012; Bruni et al., 2015).
1.2 VÍRUS PAPILOMA HUMANO E LESÕES HIPERPLÁSICAS EPITELIAIS BENIGNAS ORAIS
O vírus papiloma humano compreende uma família de DNA vírus na qual atualmente são conhecidos 170 tipos. Alguns deles são considerados de baixo risco e associados com lesões proliferativas epiteliais benignas, dentre eles, os subtipos 6/11 são os mais comuns e tem sido identificados em mais de 90% das verrugas genitais também chamadas de condiloma acuminado ou verruga venérea, assim como de outras regiões como nasal e orofaríngea (McCutcheon, 2009; de Villiers, 2013). Lesões cutâneas proliferativas induzidas por HPV são chamadas de verruga vulgar, associadas com os tipos 2, 4, 10, 40 entre outros. Os tipos 13 e 32 são associados com outra entidade que afeta cavidade oral e é bem característica de algumas regiões, a hiperplasia epitelial multifocal. Todas essas entidades são caracterizadas por crescimentos exofíticos benignos do epitélio de superfície da pele e mucosas (Eversole, 2000). Os subtipos de alto risco 16 e 18, são considerados oncogênicos e são associados com lesões displásicas intraepiteliais e carcinomas da região genital (colo uterino, pênis, vulva, vagina, anus) e orofaríngea, especificamente da tonsila. Pacientes com o vírus da imunodeficiência humana são mais suscetíveis a apresentar doenças causadas por estes subtipos (Bouvard et al., 2009). O diagnóstico das lesões genitais associadas ao HPV é principalmente baseado na aparência clínica. Os crescimentos podem ser planos, papulares ou pediculados. Quando as características são atípicas (indurada, sangrante, ulcerada), a biopsia é indicada (Workowski & Bolan, 2015). Com relação a lesões orais, a classificação existente não é tão simples, muitas
lesões não conseguem ser classificadas numa das entidades definidas pela Organização Mundial da Saúde, e existe grande sobreposição das características clínicas e histológicas, além disso a identificação do genótipo de HPV não parece ser muito útil já que os tipos 6 e 11 tem sido reportados como presentes tanto em papilomas como em condilomas (Odell, 2005).
1.3 MÉTODOS DE DETECÇÃO DO VÍRUS PAPILOMA HUMANO
Para a identificação do HPV em casos de carcinoma de cabeça e pescoço tem sido utilizados diversos métodos, sem existir ainda um consenso sobre qual o melhor. Os principais são: (1) identificação indireta pela expressão da proteína p16 por imunoistoquímica, (2) identificação do DNA ou mRNA por reação em cadeia da polimerase (PCR) e (3) hibridização in situ (ISH) para DNA e E6/E7 RNA. Dentre eles a ISH com sondas DNA é a mais utilizada devido a disponibilidade e facilidade de uso na prática clínica, embora seja difícil de interpretar e careça de completa sensibilidade. Para avaliar HPV transcricionalmente ativo, a PCR em tempo real (rt-PCR) ou a ISH para mRNA são os métodos com maior sensibilidade e especificidade, embora a técnica seja de difícil realização e a disponibilidade seja limitada. A avaliação imunoistoquímica do p16 é considerada um método para detectar HPV transcricionalmente ativo, especificamente em carcinoma de amígdala. Para confirmação da presença do HPV e para estratificação do risco é sugerida a combinação de dois métodos; identificação indireta através do p16 por imunoistoquímica, e nos casos positivos utilizar além do p16, um dos métodos diretos podendo ser a ISH ou PCR (Smeets et al., 2007; Westra, 2009; Robinson et al., 2010; Wasylyk, 2013).
1.4 p16 (INK4a)
A proteína p16 é um inibidor kinase dependente de ciclina que participa na regulação do ciclo celular. A transcrição do p16 é inibida por outra proteína reguladora do ciclo celular, o produto do gene retinoblastoma (Rb). p16 é conhecida por apresentar expressão aumentada em carcinoma de colo uterino, no qual o HPV tem iniciado o processo de transformação celular, pela expressão dos seus oncogenes virais E6 e E7. A oncoproteína E7 do HPV une-se ao Rb, regulador negativo da expressão de p16, portanto a infeção por HPV leva ao aumento da expressão nuclear de p16 (Klaes et al., 2001). Em carcinomas de colo uterino a positividade do p16 é
interpretada quando uma expressão difusa e continua no epitélio é observada, incluindo as camadas basal e parabasal, com ou sem expressão na camada mais superficial. Um padrão focal ou positividade descontinua, em células isoladas ou pequenos grupos tem sido interpretado como marcação negativa para p16 (Klaes et al., 2002). Em relação a tumores orofaríngeos, lesões com morfologia não queratinizante, positivos para HPV, tem demonstrado uma expressão forte e difusa do p16, embora não exista ainda um consenso quanto à extensão ou porcentagem de positividade de p16 que deveria ser utilizado para a identificação e classificação de tumores associados a HPV considera-se que apenas expressão extensa forte e difusa ou se for expressão parcial, maior de 75% ou maior a 50% quando mais de 25% das áreas positivas sejam áreas confluentes, seja relacionada com HPV transcricionalmente ativo (El Mofty & Lu, 2003; Lewis et al., 2010; Lewis et al., 2012). Em relação à localização, tumores laríngeos queratinizantes tem demostrado positividade para p16, sem relação com HPV. Num estudo de 76 CEC de laringe, 28% expressaram p16, em tanto que somente em 4 casos foi identificada presença de HPV por PCR ou ISH, sendo esses casos positivos para p16. Portanto, em localizações fora de orofaringe a expressão do p16 pode não ser diretamente indicativa de tumores associados ao HPV (Chernock et al., 2013).
1.5 VÍRUS EPSTEIN-BARR E CARCINOMA DE NASOFARINGE
O vírus Epstein-Barr é um gamma herpesvírus denominado vírus Herpes Humano tipo 4. Aproximadamente 90% da população adulta do mundo tem sido infectada, geralmente sendo a orofaringe o sitio da infecção primária. Esta ocorre na infância, é assintomática mas resulta na persistência do vírus ao longo da vida. Os linfócitos B e células epiteliais do trato respiratório superior são as células alvo do vírus e é neles que permanece latente para o resto da vida. Existem duas proteínas RNA não codificantes que são altamente expressas em células com infecção latente pelo EBV, EBER1 e EBER2, assim como a proteína latente de membrana 1 (LMP-1). Os EBERs tem sido considerados como responsáveis dos fenótipos malignos nas células do linfoma de Burkitt endêmico e do carcinoma de nasofaringe. Outras malignidades associadas ao EBV são Linfoma de Hodgkin, Linfoma difuso de grandes células B do idoso, Linfoma plasmablastico, Linfoma extranodal T/NK tipo nasal e outras doenças linfoproliferativas (Niedobitek et al., 2001; Delecluse et al., 2007).
O carcinoma de nasofaringe é uma doença com distribuição geográfica característica, rara na maioria dos países, mas com uma alta incidência em regiões como China, Sudeste Asiático, Norte da África, Canadá e Alaska. Afeta principalmente homens na 5ª e 6ª décadas de vida. É classificado histologicamente em queratinizante, não queratinizante e basalóide. O tipo não queratinizante compreende os subtipos diferenciado e indiferenciado. Geralmente o mesmo tumor pode ter áreas diferenciadas e indiferenciadas, sendo este último mais comum. O nome carcinoma tipo linfoepitelioma ou linfoepitelioma tem sido usado como sinônimo para este tumor devido a casos com abundante presença de linfócitos associados às ilhas neoplásicas epiteliais. Este achado é variável e o termo carcinoma linfoepitelial é melhor aplicado nos casos mais exuberantes, nos quais os linfócitos encontram-se infiltrando as ilhas tumorais e as vezes até obscurecendo a natureza epitelial do tumor (Weiss et al., 1989; Chan et al., 2005). O tipo não queratinizante é associado com o EBV em praticamente 100% dos casos. A expressão da LMP-1 (proteína latente de membrana 1) tem sido descrita como variável, em 30%-40% dos casos, geralmente num padrão focal e fraco. Pela abundancia dos RNAs EBER1 e EBER2, essas moléculas são utilizadas como alvo da hibridização in situ, método que é considerado o mais simples e confiável na detecção do vírus. É utilizada uma sonda PNA (ácido péptido nucleico) que é complementar aos dois EBER RNAs nucleares (Sarac et al., 2001; Takada et al., 2012).
No presente trabalho os autores realizaram a avaliação da expressão de HPV em casos de carcinoma de amígdala e lesões epiteliais hiperplásicas benignas orais, além da avaliação da expressão do EBV em casos de carcinoma de nasofaringe a fim de quantificar os casos positivos, classificar em subtipos de baixo e alto risco no caso do HPV e comparar a expressão entre pacientes da Guatemala e do Brasil.
2 ARTIGOS
2.1 Human papillomavirus in tonsillar squamous cell carcinomas from Guatemala and Brazil
Artigo aceito para publicação no Oral Surgery, Oral Medicine, Oral Pathology and Oral Radiology. 2016;121(4):412-418. DOI: 10.1016/j.oooo.2015.12.002 (Anexo 3)
Alicia Rumayor Piñaa, Laísa Simakawa Jimenezb, Fernanda Viviane Marianob,
Bruno Augusto Benevenuto de Andradec, Román Carlosd, Albina Altemanib,
Oslei Paes de Almeidaa
aOral Pathology Section, Department of Oral Diagnosis, Piracicaba Dental School,
University of Campinas (UNICAMP), Piracicaba, Brazil.
bDepartment of Pathology, Medical Sciences Faculty, University of Campinas
(UNICAMP), Campinas, Brazil.
cOral Pathology Section, Department of Oral Diagnosis and Pathology, School of
Dentistry, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil
dCentro Clínico de Cabeza y Cuello/Hospital Herrera Llerandi. Ciudad de Guatemala,
Guatemala.
Funding: This work was supported by the Brazilian Federal Agency for the Support and Evaluation of Graduate Education (CAPES) and the São Paulo Research Foundation (FAPESP). ∗FAPESP Process: 2013/14163-9.
Corresponding author: Alicia Rumayor Piña
Oral Pathology, Piracicaba Dental School, University of Campinas (UNICAMP), Av. Limeira 901, P.O. Box 52, 13414-903, Piracicaba, São Paulo, Brazil.
Phone: +551921065315.Fax: +551921065218. E-mail: ali84rum@hotmail.com
ABSTRACT
Objectives. A subgroup of tonsillar squamous cell carcinoma (SCC) is associated with human papillomavirus (HPV). Nevertheless, the prevalence of HPV seems to be variable in different regions and ethnic groups. There are no reports of HPV in tonsillar carcinomas in Guatemala, and data from Brazil are scarce. The aim of this study is to analyze and compare HPV presence in samples of tonsillar SCC from these countries.
Study Design. This study describes the histological features, expression of p16 by immunohistochemistry (IHC) and HPV by in situ hybridization (ISH) in 13 Guatemalan and 13 Brazilian patients.
Results. All cases of tonsillar SCC from Guatemala were positive for p16, 92% expressed HPV by ISH, and 75% corresponded to the high-risk genotype 16/18. From the Brazilian patients, only four expressed p16, and all were negative for HPV.
Conclusions. Cases from Guatemala, which were mostly nonkeratinizing SCC and originated from the crypt/reticular epithelium of the tonsil, had high-risk integrated HPV, whereas in Brazilian cases, which were mostly keratinizing SCC that originated from the surface epithelium, there was no association with HPV.
INTRODUCTION
Human papillomavirus (HPV)-related SCC is a subset of head and neck carcinomas presenting a distinct clinicopathologic profile, with increasing incidence worldwide. Besides the uterine cervix, penis, vulva, vagina, and anal region, the oropharynx is also a susceptible site for high-risk types of HPV, particularly HPV 16.1,2
Information about the incidence and prevalence of oropharyngeal carcinoma is not well documented because it is usually grouped with oral carcinoma; less reliable information is available when dealing specifically with the tonsil because tonsillar carcinomas are almost always categorized as carcinoma of the oropharynx, together with carcinomas of the base of the tongue, lingual tonsil, soft palate, uvula, and oropharynx walls.3 Reports of these entities from Guatemala are lacking; one
recent report about the burden of HPV-related cancers did not have any data for tonsillar cancer.4 Data from Brazil are unclear because of the differences in the
categorization of subsites. When reported only as oropharyngeal carcinoma, percentages varied from 16% to 23%, of all head and neck carcinomas.3,5 In studies
specifically reporting tumors from the tonsil, percentages varied from 4% to 13% in three different cities from Brazil to 30% in Central and South America.6-9
HPV-associated oropharyngeal SCCs occur mainly in the tonsil of male patients, showing an increased survival rate compared with HPV-negative cases. This subset of carcinomas also show histologic and molecular features different from conventional SCCs, as these usually are nonkeratinizing, strongly express p16, and show a higher proliferation index and lower p53 expression. Some authors have stated
that p16, by itself, is a good marker for favorable prognosis, regardless of HPV status.10,11
It is probable that geographic/genetic differences exist in relation to the role of HPV in oropharyngeal carcinomas, and wide variation in prevalence has been observed among different regions and countries. In North America and some European countries, HPV seems to be the primary cause of tonsillar carcinoma. In contrast, a study from China reported absence of HPV in their samples of 16 tonsillar SCC, attributing these results to cultural and behavioral differences.12-14
In the United States, the incidence of tonsillar carcinomas associated with HPV is highly variable, with percentages ranging from 22% to 90.9%, but in most reports, values are between 35% and 65%. In a worldwide systematic review of 969 oropharyngeal SCC cases, HPV was identified in 36%, with higher prevalence in North America (47%) and Asia (46%).15-17 In a recent Mexican study, which included 43 head
and neck SCCs, 42% affected the oropharynx, and 35% the oral cavity. Of the oropharyngeal SCCs, 61% were associated with HPV when analyzed by polymerase chain reaction (PCR); an interesting finding was that risk habits for classic oral SCC, such as smoking, were less common in the HPV-positive group compared with the HPV-negative cases.18 A study of head and neck SCCs in Brazil, including various sites
(pyriform sinus, larynx, tonsil, mouth and maxillary sinus), showed a low prevalence of HPV by PCR (11%), and two out of six cases of tonsillar SCC were positive for HPV.6
In recent meta-analysis, HPV prevalence in oropharyngeal SCC was low in South and Central Americas; however, data from these regions were limited.13-15 The
association of known risk factors, such as tobacco and alcohol, with oropharyngeal SCC has been found to be minimal in HPV-positive cases, whereas higher levels of consumption were reported in HPV-negative patients.19,20 The aim of this study was to
determine the presence of HPV in tonsillar SCC in patients from Guatemala and Brazil. MATERIALS AND METHODS
Formalin-fixed, paraffin-embedded tissue blocks of 13 tonsillar SCC were retrieved from the Centro Clínico de Cabeza y Cuello, Guatemala, and 13 from the Pathology Department, UNICAMP, Brazil. The clinical data for all cases were obtained from medical records. Smoking and alcohol drinking were measured and classified according to Ribeiro et al.21
All cases were histologically classified as keratinizing (K) and nonkeratinizing (NK) SCCs, and those with overlapping features were considered NK-type SCC presenting focal areas of squamous maturation or keratinization (NK/focal K).
Immunohistochemistry (IHC) for p16INK4a (CINtec Histology Kit, MTM
laboratories, Heidelberg, Germany) was performed in 3-μm sections, according to the manufacturer´s protocol. All cases were classified as positive or negative, the former when more than 80% of tumor cells presented strong, diffuse, nuclear, and cytoplasmic staining.22 HPV detection was performed by using ISH, with a wide spectrum probe
(Y1404; Dako, Carpinteria, CA) which includes genotypes 6, 11, 16, 18, 31, 33, 35, 45, 51, and 52, and with a 16/18-specific probe (Y1412; Dako, Carpinteria, CA). Then, 5-μm sections were deparaffinized and subjected to epitope retrieval in citrate buffer, hybridized, and labeled by the Catalyzed Signal Amplification System (K0620; Dako, Carpinteria, CA). For IHC and ISH, a case of carcinoma of the uterine cervix was used as positive control and a case of a known HPV-negative well-differentiated tongue SCC was used as negative control. The presence of dark brown diffuse or punctate (dots) nuclear signal within the tumor cells was considered positive.1
This study was approved by the Ethics Committee in Research of the Piracicaba Dental School - University of Campinas with the registry number 072/2014 and was certified that it complied with the recommendations of the National Health Council - Ministry of Health of Brazil for research in human patients.
RESULTS
All cases studied were primary tumors from the tonsil, except three cases and one case among the samples from Guatemala and Brazil, respectively, which corresponded to lymph node metastases from primary tumors of the tonsil. Tonsillar SCC from Guatemala included 12 males and one female, with a mean age of 61 years (range 43-78 years), whereas cases from Brazil included 12 males and 1 female, with a mean age of 56 years (range 43-76 years). With regard to classic head and neck SCC risk factors, among the Guatemalan patients, 31% reported being former or current smokers and drinkers, and in the Brazilian group, the percentage rose to 85%.
Most tonsillar SCCs from Guatemala were NK (69%), three (23%) were NK/focal K, and only one was K (Figure 1). NK carcinomas seemed to originate from the tonsillar crypt epithelium (reticular epithelium), four of them showing a
lymphoepithelial/nasopharyngeal-like morphology. With regard to the Brazilian cases, most were K (61%), and 31% were NK (one showing lymphoepithelial/nasopharyngeal-like morphology), and one was NK/focal K.
All 13 cases from Guatemala were strongly positive for p16 in the cytoplasm and nuclei, whereas only four out of 13 from Brazil were considered positive: one NK had lymphoepithelial morphology, and three were K. All except one case of tonsillar SCC from Guatemala were positive for wide-spectrum probe for HPV, and nine cases also expressed high-risk HPV, type 16/18. Expression was mainly as a nuclear punctuate pattern (Figure 2). The 13 tonsillar SCC cases from Brazil were negative for HPV, by wide- spectrum or specific high-risk probe. Among the metastatic lymph nodes from Guatemala, two were NK and one showed focal keratinization, cystic morphology was evident in two cases. All three cases expressed wide-spectrum and 16/18 HPV in a dot pattern (Figure 3). Summarized results from Guatemala cases are shown in Table I and those from Brazil in Table II.
DISCUSSION
It is interesting to note that the mean age of patients from Guatemala and Brazil was around 60 years; in contrast, ages lower than 60 years seemed to be a trend for SCC of the tonsil, positive or negative for HPV. A study in Brazil, including SCC, papillomatous epithelial hyperplasia, and papillomas from sites such as the mouth and the tonsil, reported that in HPV-positive cases, the mean age was 51 years, whereas HPV-negative patients were two decades older.23
HPV-associated SCC of the oropharynx is attributed mainly to sexual behavior, but it is not known whether this factor alone explains the high prevalence in Guatemala and the low prevalence in Brazil. One point to consider is that in this study, most of the Brazilian patients were smokers and drinkers, whereas the opposite was observed in the Guatemalan group. Ribeiro et al. recently reported a low HPV prevalence in a population of patients in central Europe with head and neck SCC, who consumed tobacco and alcohol.21 Similarly Soares et al. also observed that in
HPV-negative cases, the level of tobacco consumption was significantly higher.19 Our results
are compatible with previous reports, showing that in Brazil, the main causes of tonsillar carcinomas are alcohol and tobacco.13,24
It has been suggested that NK tonsillar carcinoma originates from the reticular crypt epithelium, presenting higher susceptibility for HPV infection. Tumors
from the tonsillar surface epithelium are more often keratinizing and HPV-negative. 25-28 These observations were confirmed in the present study, as most cases from
Guatemala were of the NK type and HPV-positive, and the opposite was true of the Brazilian cases, indicating also that the latter probably had originated mainly from the superficial epithelium of the tonsil.
Oropharyngeal SCC expressing HPV is associated with a better prognosis, although histologically it is more undifferentiated and aggressive than those that are HPV- negative and usually keratinized. In oropharyngeal carcinoma, p16 is accepted as a simple and good marker to indicate HPV infection and prognosis, and it can also be used as surrogate marker in metastatic tumors when determining the primary localization.12,29 Our sample from Guatemala included three cases of metastatic lymph
nodes of tonsillar SCC, and all were positive for p16 and HPV, confirming that this information can be helpful when dealing with occult primary tumors.
The methodology used to detect HPV in oropharyngeal carcinomas and the criteria used to determine positivity contribute to the differences in incidence found in many reports. Nevertheless, some indirect parameters are well accepted, such as NK morphology and strong nuclear and cytoplasmic expression of p16. Detection of HPV by ISH is an additional specific method, which permits confirmation of high-risk HPV genotypes, particularly types 16 and 18. PCR is frequently used and is considered by many as the best method because of its high sensitivity, but it must be kept in mind that it may produce “false-positive” results. In tonsillar SCC, histopathologic pattern and strong positivity for p16 are still good indicators of high-risk HPV infection, and this can be confirmed by detection of HPV with ISH, as done in this study.30,31
CONCLUSIONS
The results of this study show that all tonsillar SCCs in patients from Guatemala were HPV-positive, and most cases from Brazil were negative, suggesting that HPV is not associated with the disease in our sample from Brazil and that other factors need to be studied to better understand the higher susceptibility for HPV infection in the Guatemalan patients. Also, as histologic appearance was completely different between both groups, suggesting differences in site of origin (crypt vs surface), and because this different subtypes of SCC has different oncologic pathways, we were analyzing two separate biologic entities and confirming that NK-type tonsillar SCC has higher integrated HPV.
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5. Alvarenga Lde M, Ruiz MT, Pavarino-Bertelli EC, Ruback MJ, Maniglia JV, Goloni-Bertollo M. Epidemiologic evaluation of head and neck patients in a university hospital of Northwestern São Paulo State. Braz J Otorhinolaryngol. 2008;741:68-73.
6. Miguel RE, Villa LL, Cordeiro AC, Prado JC, Sobrinho JS, Kowalski LP. Low prevalence of human papillomavirus in a geographic region with a high incidence of head and neck cancer. Am J Surg. 1998;176:428-429.
7. Silva PSL, Leão VML, Scarpel RD. Characterizing the population with mouth and orofaringe cancer, attended in the sector of head and neck in a referral hospital in Salvador City – BA. Rev. CEFAC [online]. 2009;11:441-447. Available at: http://dx.doi.org/10.1590/S1516-18462009000700020.
8. Melo LC, Silva MC, Bernardo JM, Marques EB, Leite IC. Epidemiological profile of incident cases of oral and pharyngeal cancer. Rev Gaúcha Odontol. 2010;58:351-355.
9. de Camargo Cancela M, de Souza DL, Curado MP. International incidence of oropharyngeal cancer: a population-based study. Oral Oncol. 2012;48:484-490. 10. Lewis JS, Thorstad WL, Chernock RD, et al. p16 Positive oropharyngeal
squamous cell carcinoma: an entity with a favorable prognosis regardless of tumor HPV status. Am J Surg Pathol. 2010;34:1088-1096.
11. Chaturvedi AK. Epidemiology and Clinical Aspects of HPV in Head and Neck Cancers. Head Neck Pathol. 2012;6:S16–S24.
12. Li W, Thompson CH, Xin D, et al. Absence of Human Papillomavirus in Tonsillar Squamous Cell Carcinomas from Chinese Patients. Am J Pathol. 2003;163:2185–2189.
13. Marur S, D´Souza G, Westra WH, Forastiere AA. HPV-associated head and neck cancer: a virus-related cancer epidemic. Lancet Oncol. 2010;11:781-789. 14. Ndiaye C, Mena M, Alemany L, et al. HPV DNA, E6/E7 mRNA, and p16INK4a detection in head and neck cancers: a systematic review and meta-analysis. Lancet Oncol. 2014;15:1319-1331.
15. Kreimer AR, Clifford GM, Boyle P, Franceschi S. Human papillomavirus types in head and neck squamous cell carcinomas worldwide: a systematic review. Cancer Epidemiol Biomarkers Prev. 2005;14:467-475.
16. El Mofty SK, Lu DW. Prevalence of Human Papillomavirus Type 16 DNA in Squamous Cell Carcinoma of the Palatine Tonsil, and Not the Oral Cavity, in Young Patients. A Distinct Clinicopathologic and Molecular Disease Entity. Am J Surg Pathol. 2003;27:1463–1470.
17. Rusan M, Ovesen T. Worldwide Prevalence of Human Papillomavirus in Tonsillar Squamous Cell Carcinoma and Tumor-Free Tonsillar Tissue. Otolaryngology. 2012;S2:001.
18. Ibieta-Zarco BR, Carrillo-García A, Ponce-de-León-Rosales S, Flores-Miranda MM, Mohar A, Lizano M. Frequency and genotype distribution of multiple human papillomavirus infections in cancer of the head and neck in a Mexican population. Oral Surg Oral Med Oral Pathol Oral Radiol. 2012;114:350-357. 19. Soares GR, Demathé A, Mattar NJ, Biasoli ER, Miyahara GI. Absence of HPV
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TABLES
Table I. Clinical and histopathologic features, p16 (immunohistochemistry) and human papillomavirus (in situ hybridization) expression in 13 cases of tonsillar squamous cell carcinoma of Guatemalan patients
Case Age (years)/ gender
Tumor site Smoking Alcohol drinking Histologic classification p16 HPV WS HPV 16/18
1 74/M Tonsil Never Never NK + + +
2 51/M Tonsil Former Current NK + + Neg
3 73/M Tonsil Never Never NK + + +
4 78/M Cervical
lymph node*
Never Never NK + + +
5 66/M Tonsil Current Current K + + Neg
6 49/M Cervical
lymph node*
Never Never NK/focal K + + +
7 66/M Tonsil Never Never NK + + +
8 70/M Tonsil Never Never NK + + Neg
9 63/M Cervical
lymph node*
Never Never NK + + +
10 68/F Tonsil Current Current NK + + +
11 43/M Tonsil Current Never NK/focal K + Neg Neg
12 51/M Tonsil Never Never NK + + +
13 48/M Tonsil Never Never NK/focal K + + +
M, male; F, female; K, keratinizing; NK, nonkeratinizing; WS, wide spectrum; +, positive; Neg, negative.
papillomavirus (in situ hybridization) expression in 13 cases of tonsillar squamous cell carcinoma of Brazilian patients
Case Age (years)/ gender
Tumor site Smoking Alcohol drinking Histologic classification p16 HPV WS HPV 16/18
1 63/M Tonsil Current Current K + Neg Neg
2 53/M Cervical
lymph node*
Never Never NK Neg Neg Neg
3 60/F Tonsil Current Current K + Neg Neg
4 76/M Tonsil Current Current K Neg Neg Neg
5 53/M Tonsil Current Current NK Neg Neg Neg
6 54/M Tonsil Current Current K Neg Neg Neg
7 63/M Tonsil Never Never K Neg Neg Neg
8 43/M Tonsil Current Current NK + Neg Neg
9 59/M Tonsil Current Current K + Neg Neg
10 61/M Tonsil Current Current K Neg Neg Neg
11 51/M Tonsil Current Current NK Neg Neg Neg
12 45/M Tonsil Current Current NK/focal K Neg Neg Neg
13 52/M Tonsil Current Current K Neg Neg Neg
M, male; F, female; K, keratinizing; NK, nonkeratinizing; WS, wide spectrum; +, positive; Neg, negative.
Fig. 1. Histologic features of tonsillar squamous cell carcinoma (SCC). A, Keratinizing SCC showing eosinophilic large squamous pleomorphic cells, and evident areas of keratin. B, Nonkeratinizing SCC. Nest of small basophilic undifferentiated cells with comedo-necrosis. C, Nonkeratinizing SCC with focal areas of keratin maturation. D, Nonkeratinizing SCC, showing lymphoepithelial/nasopharyngeal-like features (H&E, x200).
Fig. 2. p16 and human papillomavirus (HPV) expression in tonsillar squamous cell carcinoma (SCC). A, Low-power view of a nonkeratinizing lymphoepithelial-like SCC. The surface stratified squamous epithelium can be seen at the upper right corner (H&E, x100). B, Strong expression of p16 restricted to the carcinomatous areas (immunohistochemistry [IHC], x100). C, Nonkeratinizing SCC formed by basophilic cells with indistinct borders and scanty cytoplasm (H&E, x200). D, Strong, diffuse, and confluent nucleocytoplasmic expression of p16 (IHC, x200). E, Wide-spectrum HPV probe showing positivity in a dot-like pattern (in situ hybridization [ISH], x1000). F, HPV probe types 16/18 showing a dot-like positive pattern (ISH x1000).
Fig. 3. Metastatic lymph node showing nonkeratinizing squamous cell carcinoma (SCC) from primary of the tonsil. A and C, Nonkeratinizing SCC with papillary and cystic features, respectively (x50). B and D, Strong and diffuse p16 expression in both cases (immunohistochemistry [IHC], x50). E, Wide-spectrum human papillomavirus (HPV) positivity in a punctuate/dot-like pattern inside the nucleus of tumoral cells (in situ hybridization [ISH], x1000). F, High-risk HPV, type 16/18 showing dot-like positivity (ISH, x1000).
2.2 Benign epithelial oral lesions related to human papillomavirus, overlaps and difficulties in diagnosis
Alicia Rumayor Piña, DDS, MSc1, Felipe Paiva Fonseca, DDS, PhD1, Flávia
Sirotheau Corrêa Pontes, DDS, PhD2, Fábio Ramôa Pires, DDS, PhD3, Mario José
Romañach, DDs, PhD4, Roman Carlos, DDS5, Oslei Paes de Almeida, DDS, PhD1 1Oral Pathology Section, Department of Oral Diagnosis, Piracicaba Dental School,
University of Campinas (UNICAMP). Piracicaba, Brazil.
2Service of Oral Pathology - João de Barros Barreto University Hospital - Federal
University of Pará. Belém, Brazil.
3Oral Pathology, Department of Surgery and Diagnosis, Dental School, State
University of Rio de Janeiro. Rio de Janeiro/RJ, Brasil.
4Oral Pathology Section, Department of Oral Diagnosis and Pathology, School of
Dentistry, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil.
5Centro Clínico de Cabeza y Cuello/Hospital Herrera Llerandi. Ciudad de Guatemala,
Guatemala.
Corresponding author: Alicia Rumayor Piña
Oral Pathology, Piracicaba Dental School, University of Campinas (UNICAMP), Av. Limeira 901, P.O. Box 52, 13414-903, Piracicaba, São Paulo, Brazil.
Phone: +551921065315.Fax: +551921065218. E-mail: ali84rum@hotmail.com ABSTRACT
Benign epithelial oral lesions related to human papillomavirus are a group characterized microscopically by acanthosis, papillomatosis and koilocytosis. Overlapping features are seen between squamous papilloma, condyloma acuminatum, multifocal epithelial hyperplasia and verruca vulgaris and confusion is generated regarding to terminology and association with human papillomavirus. We analyzed 87 oral lesions clinically suggestive of being HPV-related. In situ hybridization revealed the presence of human papillomavirus in 24% of the cases, from these, 67% showed to be HPV 6/11 positive. Oral condyloma acuminatum and flat condyloma were the lesions more associated with HPV. Proliferation marker Ki67 revealed a distinct and abnormal pattern in HPV associated lesions. The most prevalent lesion was squamous papilloma, which do not show association with HPV, this group deserve further studies to better clarify its etiology.
Key words: Human papillomavirus. Oral lesions. Condyloma. Papilloma. INTRODUCTION
The World Health Organization (WHO) recognizes three oral epithelial lesions related to human papillomavirus (HPV), squamous papilloma (SP)/verruca vulgaris (VV), condyloma acuminatum (CA) and focal epithelial hyperplasia (FEH). SP and VV are grouped together and defined as hyperplastic wart-like proliferations of the oral epithelium. When HPV antigens are detected in a papilloma, they consider it the intraoral counterpart of cutaneous VV, also histologically resembling it. Most papillomas seems to be negative for HPV, showing variable histopathological features; besides acanthosis, the surface can be either papillary or flat, with no evidences of koilocytes or keratohyaline granules. Squamous papilloma positive for HPV (oral verruca vulgaris) and negative are clinically indistinguishable. These observations made by the WHO were based in old studies that used immunohistochemistry (IHC) for the detection of HPV antigens, so HPV subtypes could not be identified (Syrjänen et al., 1984; Welch et al., 1986; Garlick & Taichman, 1991; Odell, 2005). Recent studies show that HPV is detected in approximately half of the papillomas, with the subtypes 6 and 11 as the most prevalent, while HPV subtypes 2 and 57 in vermilion border verruca vulgaris. The existence of a true intraoral verruca vulgaris is doubtful (Eversole et al., 1987; Syrjänen, 2003). Oral CA corresponds to the counterpart of genital condyloma, usually positive for HPV 6, 11 and less commonly for HPV 16 and 18 (Anderson et al., 2003; Odell, 2005; Tomson et al., 2011; Grce & Stipetić, 2014). Focal epithelial hyperplasia has well established features with multiple lesions associated to HPV 13 and 32, affecting specific groups of individuals in a few regions of the world, particularly Indians of Latin America and Eskimos (Carlos & Sedano, 1994; Durso et al., 2005; Said et al., 2013). Recently, it was described a severe oral dysplasia associated with high-risk HPV, histologically similar to lesions of the uterine cervix, that was called HPV-associated oral intraepithelial neoplasia or koilocytic dysplasia (Fornatora et al., 1996; Woo et al., 2013; McCord et al., 2013). Clinical and histopathological features are supposed to be sufficient to differentiate these groups of oral epithelial lesions; however, in an oral pathology daily routine, this differentiation can be difficult as misuse of terms and overlapping features generate confusion. We herein describe and discuss the clinical, histopathological features and HPV presence
of 87 oral benign epithelial hyperplastic lesions and review the literature to better clarify this group of oral mucosa lesions.
MATERIALS AND METHODS
We retrieved from our files 87 oral lesions that were clinically suggestive of being HPV-related. All of them had available clinical image, H&E slide and paraffin blocks with enough tissue for HPV detection by in situ hybridization (ISH) and immunohistochemical reactions. Clinical and histological diagnoses were made independently by 4 oral pathologists, first considering only clinical images and then representative microscopic aspects. Orientation was given to use the criteria defined by the WHO including the diagnoses of squamous papilloma, verruca vulgaris, condyloma acuminatum and focal epithelial hyperplasia (Odell, 2005). When there was not a consensus, the decision of the final diagnosis was made by the most experienced pathologist. Then, a third classification was made using both histological and clinical diagnosis, considering only concordant diagnoses. In addition, for histological comparison, 13 cases of genital condyloma acuminatum previously known to be HPV 6/11 positive were included.
In the 87 cases of oral lesions, after deparaffinization and antigen retrieval in citrate buffer pH 6.0, ISH was performed overnight at 37°C in 5µm sections using a wide spectrum probe that includes genotypes 6, 11, 16, 18, 31, 33, 35, 39, 45, 51 and 52, and 2 specific probes, one for HPV 6/11 and other for 16/18 (Y1404, Y1411, Y1412 respectively; Dako, Carpinteria, CA), using for visualization the Catalyzed Signal Amplification System (K0620; Dako, Carpinteria, CA). Cases of cervical and oral tongue squamous cell carcinoma were used as positive and negative controls. The presence of a complete nuclear (episomal) or punctate/dots (integrated) dark brown signals within the epithelial cells were considered positive (Evans et al., 2002). Immunohistochemistry (IHC) for Ki67 (MIB-1, 1:100, Dako) and p16INK4a (CINtec Histology Kit, MTM laboratories) was also performed in 3µm sections of all the cases and one case, respectively.
RESULTS
The total sample consisted of 47 males (54%) and 40 females (46%) with a mean age of 41 years. Solitary lesions were seen in 73 cases and multiple lesions in
14. Affected sites included tongue (24), palate (22), labial mucosa (17), lip vermilion (13), gingiva (7), buccal mucosa (6) and floor of the mouth (4).
Considering only the clinical features, the 87 oral hyperplastic epithelial lesions were classified as squamous papilloma (57), verruca vulgaris (12), condyloma acuminatum (11), focal epithelial hyerplasia (5) and 2 cases in which there was no consensus on whether they were condylomas or carcinomas. When reviewing only histopathology, 55 cases were diagnosed as papilloma, 14 as condyloma; three of them were diagnosed as flat condyloma, and one showed evidences of mild/moderate dysplasia restricted to focal areas of the basal layer, 12 as verruca vulgaris, 2 as focal epithelial hyperplasia, 2 as fibrous hyperplasia and one each of severe epithelial dysplasia and exophytic carcinoma. The clinical and microscopical diagnoses were concordant in 78 of the 87 cases, 54 were papillomas, 12 verruca vulgaris, 10 condylomas and 2 focal epithelial hyperplasias.
The 54 cases with concordant diagnosis of papilloma were localized mostly in soft palate, tongue and labial mucosa. Patients were mainly females with mean age of 43 years. Clinically most were solitary small lesions, sessile or pedunculated, surface could be papillary, micro papillary, digitiform, blunt, rounded or cauliflower-like, normal mucosa or white in color. Histologically, most of the papillomas consisted of thin exophytic projections of squamous epithelium with a central core of connective tissue, while the surface showed areas of para and orthokeratin. Three cases had a thick layer or hyperorthokeratin and prominent keratohyaline granules. Cells suggestive of koilocytes were seen in 18% of the papillomas.
All 12 cases classified clinically and histologically as VV involved the lip vermilion, 8 the upper lip, 2 the lower and 2 the commissure. Affected patients were mainly between the 1st and 3rd decade of life with a mean age of 22 years. Clinically, solitary small slightly elevated papules were seen, presenting a whitish rough surface with evident hard-pointed projections. Microscopically, VV showed epithelial papillary projections, with a prominent layer of orthokeratin and abundant keratohyaline granules. Groups of cells with clear perinuclear halo and irregular nuclear membranes suggestive of koilocytes were seen in 83% of the cases, mainly in the upper portion of the spinous stratum and in the grooves between the papillary projections.
The 10 cases classified as condyloma affected mainly the labial mucosa and tongue of males with a mean age of 45 years. Clinically, multiple rounded sessile large growths, generally normal-colored, with short blunt-ended projections and a mulberry-like surface were seen. In 70% of the cases, multiple clustered lesions were present. Histologically, broad papillary projections of squamous epithelium with small cores of connective tissue were seen. The papillae were wide and bulbous, and the surface hyperkeratinized. In 90% of the cases, a thick spinous layer was evident, containing in its most upper portion abundant cells with characteristics of koilocytes (Fig. 1).
The 2 cases of focal epithelial hyperplasia showed multiple flat normal-colored smooth coalescent lesions; giving the typical cobblestone appearance involving the labial and buccal mucosa of an 8 and a 11-year-old boys. Microscopically these cases showed areas of marked acanthosis with mixed areas of para and orthokeratin, slightly papillary surface and koilocytes. Rete processes or connective tissue cores were not evident.
Finally, 9 cases had no agreement when considering clinical and histological diagnosis together. From these, 3 had clinical features of focal epithelial hyperplasia but were flat condyloma (2) and papilloma (1) histologically. Another 3 cases had clinical features of papilloma but histologically were diagnosed as fibrous hyperplasia (2) and flat condyloma (1) (Fig. 2). Two cases were large papillary lesions resembling condyloma and also suggesting carcinoma clinically, histologically one of them showed to be an exophytic SCC and the other a condyloma with focal dysplasia of the basal layer (Fig. 3). One case was diagnosed as condyloma clinically, but histologically it was a severe intraepithelial dysplasia. Koilocytes were seen in 55% of these discordant cases, which histologically were the 3 flat condylomas, the condyloma with basal dysplasia and the only case of severe intraepithelial dysplasia.
The 13 cases of genital and anal condylomas affected the penis (5), anus (5) and vulva (3). Histologically, a papillary proliferation of squamous epithelium was seen, with variable amounts of para or orthokeratin. All of them presented easily identifiable koilocytes, localized mainly above the spinous layer and characterized by a perinuclear cytoplasmic vacuolization and variable nuclear alterations irregular nuclear contours, hyperchromatism and pyknosis (Fig. 4).
HPV detection by ISH
From the 87 cases of hyperplastic epithelial oral lesions, 21 (24%) were positive for the wide-spectrum HPV probe. From these, 14 (67%) were positive for the 6/11 specific probe, only one for the subtypes 16/18, and 6 (29%) were negative for both probes. The 14 cases positive for probe 6/11 corresponded to 7 condylomas, 3 papillomas, 3 discordant cases (histologically 2 flat condylomas and 1 condyloma with basal dysplasia) and 1 focal epithelial hyperplasia. The 6 negative cases for the two specific probes were 3 lip vermilion VV, 2 CA and 1 discordant case (flat condyloma histologically). The only case positive for the 16/18 specific probe was a 60-year-old HIV-positive man, showing multiple plaques involving the left buccal mucosa and soft palate, clinically compatible with the diagnosis of condyloma that microscopically revealed severe dysplasia, characterized by a corrugated surface of eosinophilic parakeratin, abundant apoptotic bodies, multinucleated pleomorphic cells, karyorrhexis, figures of aberrant mitoses and koilocytes. In this case, besides ISH for HPV, and IHC for Ki67, we also performed p16, showing positivity in almost all the epithelial cells for the former and a strong nucleo-cytoplasmic diffuse expression for the latter. Final diagnosis of this case was oral intraepithelial neoplasia (koilocytic dysplasia) as described previously (Fornatora et al., 1996; Woo et al., 2013). From the 66 cases negative for the HPV probes used, 51 (77%) had concordant diagnosis of papilloma, 9 as VV, 4 were discordant cases and 1 each CA and FEH. Cells suggestive of koilocytes were seen in 15 (23%) of these cases, mostly in papillomas and VV. Ki67 immunoexpression
Ki67 highlighted proliferating cells restricted to the basal layer in most of the 87 cases studied. However, in 28 cases (32%) a different expression pattern was identified, from these, 68% were HPV positive. In 22 cases, besides the basal layer positivity, focal groups of cells expressed Ki67, mainly near the surface. These cases were 9 VV, 8 CA and 5 papillomas, from these, 64% were HPV positive. In 6 cases positivity was observed in the whole thickness of the epithelium, and corresponded to 4 discordant cases (histologically 3 flat condylomas and the intraepithelial dysplasia) and 2 condylomas. From these cases, 83% were HPV positive. This abnormal pattern of proliferation was also seen in 92% of the genital condylomas, positivity was seen in
the whole thickness of the epithelium in 54% of the cases, while in 38% it was observed in focal areas of the upper half (Fig. 5).
DISCUSSION
HPV subtypes 6 and 11 are the most commonly associated with oral epithelial hyperplastic lesions (Syrjänen, 2003). In fact, we found these types in 67% of the 21 HPV-positive lesions. In papillomas, these subtypes have been reported in 4% to 35% of the cases, we found them in 5% of our sample of 54 papillomas, in contrast to HPV-negative papillomas, these cases showed koilocytes and an abnormal expression of Ki67. Regarding to clinical features, both groups are indistinguishable. It should also be considered here the possibility of an eventual case associated with an HPV subtype not included in the wide spectrum probe. In oral condylomas types 6/11 have been found in 85% to 100% of the cases (Eversole et al., 1987; Eversole & Laipis, 1988; Zeuss et al., 1991; Miller et al., 1991). We found these subtypes in 70% of our 10 lesions diagnosed as condylomas. These cases showed clinical features as larger size (>15mm), multiplicity and clustering. Histologically, short blunted/rounded epithelial projections, bulbous processes, and abundant koilocytes in the upper layers of epithelial crevices were seen, as reported previously (Praetorius, 1997; Anderson et al., 2003). As oral papillomas positive for HPV share the same virus subtypes with condylomas (HPV 6/11), this raises the possibility whether these lesions belong to the same spectrum with different clinical presentation. In fact, in the genitalia, papilloma and condyloma are usually grouped as condyloma. Oral condyloma is considered to be acquired mainly from oral-genital contact, although autoinoculation or maternal transmission cannot be excluded, and possibly the same can be said for the group of HPV-positive papillomas (Syrjänen, 2003).
Koilocytes have been reported in 100% of oral condylomas and 45% of papillomas, similar to our findings (Eversole, 1988). In cervical lesions and genital warts, koilocytes are usually abundant and evident, while in oral mucosa these viral altered cells are difficult to identify with certainty, because they can be confused with vacuolated epithelial cells, which are common in the oral epithelium. However, strict criteria for koilocyte identification should be applied, besides the perinuclear clear halo, nuclear enlargement, hyperchromasia, irregular nuclear outlines and multilobation
should be searched (Chang et al., 1991; Varnai et al., 2009; Cho et al., 2005; Woo et al., 2013).
The HPV high-risk types 16/18, which are the most common found in cervical and anogenital carcinomas are rare in oral benign hyperplastic lesions. These subtypes have been found in dysplastic and malignant oral lesions, yet in few cases. In a study of 77 cases of oral dysplasia, these subtypes were found in 9%; accordingly, we did not found these subtypes in any case of benign hyperplastic lesion. The only one case of our sample that showed positivity for subtypes 16/18 was histologically a severe epithelial dysplasia. Studies in oral carcinoma are controversial as PCR is usually leading to high number of false positive cases (McCord et al., 2013; Walline et al., 2013; Grce & Stipetić, 2014). Therefore, HPV 16/18 are rarely found in the oral mucosa, indicating a possible mechanism of natural resistance of the oral epithelial cells to these aggressive HPV types. This is an explanation for the low association of oral cancer and HPV, different from the cervix and oropharynx (Herrero et al., 2003; Forman et al., 2012; Cubie, 2013).
Regarding to our 5 cases clinically suggestive of FEH, we expected negativity for the probes used, because these lesions are known to be associated with HPV 13 and 32, whose probes are not commercially available. Nevertheless, one case affecting an 11-year-old boy was positive for the HPV wide spectrum probe and for the 6/11 specific probe. It is interesting that some studies demonstrated that these HPV types could eventually be associated with FEH (Padayachee et al., 1991; Syrjänen et al., 1987; Khanal et al., 2015). The term FEH as used by the WHO is misleading because this entity is typically formed by multiple lesions that involve mainly the lips but also tongue and buccal mucosa, therefore the term multifocal seems more appropriate as suggested by Carlos & Sedano in 1994 (Carlos & Sedano, 1994).
Ki67 showed to be interesting to indicate association of oral benign epithelial hyperplasias with HPV, as almost 70% of the cases showing positivity non-restricted to the basal layer i.e. positive cells also near the surface or in the whole thickness of the epithelium, resulted to be positive for HPV. It is important to consider that these lesions should be distinguished from epithelial dysplasias not associated with HPV.
In conclusion, as the wide spectrum probe that we used covers 11 genotypes out of 170 that are actually known, we did not detect eventual cases that
could be positive for other types as cited above for FEH and VV (Li et al., 2011; de Villiers, 2013; Cubie, 2013). In fact, we did not find reports in the English literature considering the presence of other HPV subtypes not covered by the wide spectrum probe used in this work in oral benign epithelial hyperplasias. As cited, most of the positive cases for the wide spectrum probe were also positive for 6/11 and diagnosed as condylomas. Most oral papillomas were negative for HPV; the low number of positive cases showed koilocytosis and an abnormal Ki67. One point that deserves further considerations is the lesions diagnosed as papillomas that can be positive or negative for HPV, as the etiology seems to be different. Although high discrepancies are found when using methods as ISH or PCR, as the latter possess a higher sensitivity but lower specificity, studies using PCR shows higher percentages of positivity, nevertheless even with this method, there is a significant group of papillomas negative for HPV (Ward et al., 1995). This group deserves further studies to better clarify its etiology.
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