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Prolifererande verrukös leukoplaki, PVL, är en ovanlig sjukdom som yttrar sig genom multipla, vita förändringar i munslemhinnan som recidiverar och med tiden riskerar att utvecklas till cancer. Diagnosen kan endast ställas kliniskt när sjukdomsförloppet pågått under lång tid och det finns heller inga effektiva behandlingsmetoder. Etiologin till sjukdomen är okänd, men idag vet man att det finns ett samband mellan cancerutveckling och vissa virustyper.Syftet med studien är att undersöka om det går att påvisa humant papillom virus, HPV, i vävnadsprover från patienter som kan misstänkas ha PVL. Urvalet bestod av 11 patientfall, där vävnadsprover från biobanken vid avdelningen för oral patologi, Malmö högskola, samlats in och studerats med kromogen in situ hybridisering. Resultaten visade en högre förekomst av HPV i biopsier tagna i det senare skedet av sjukdomen jämfört med tidigare biopsier. En specifik HPV-genotyp kunde dock inte identifieras. I denna studie har det inte varit möjligt att påvisa HPV med in situ hybridisering, i ett tidigt staddium av PVL, som en möjlig indikator på sjukdomsutveckling. / Proliferative verrucous leukoplakia, PVL, is a rare disease that is characterized by multiple and recurrent white lesions in the oral epithelium which over time may develop into cancer. The diagnosis can only be made by the clinician when the disease has progressed for a long time. There is no effective treatment. The etiology of the disease is unknown but today it is known that there is a link between cancer and certain viruses.The purpose of this study is to investigate whether it is possible to detect human papilloma virus, HPV, in tissue samples from patients who possibly are affected by PVL.The selected samples consisted of 11 cases in which tissue samples from the biobank at the Department of Oral Pathology at Malmö University, were collected and HPV was detected by chromogenic in situ hybridization. The results showed a higher incidence of HPV in biopsies taken at a later stage of the disease compared with previous biopsies. However, a specific HPV genotype could not be identified.In this study, it has not been possible to demonstrate HPV with in situ hybridization at an early state of PVL as a putative indicator of disease development

L’accouchement prématuré a été récemment associé à la persistance du virus du papillome humain de type 16 (VPH-16) en grossesse. Il demeure toutefois difficile de savoir si cette association est causale et d’en expliquer les mécanismes biologiques potentiels. Afin de mieux caractériser cette association, nous avons étudié l'association entre la charge virale du VPH-16 en grossesse et l’accouchement prématuré. Les données de 48 femmes enceintes qui étaient positives pour le VPH-16 dans la cohorte HERITAGE ont été analysées avec un modèle de régression logistique, où la confusion a été ajustée avec scores de propension et pondération par l’inverse de probabilité de traitement. La charge virale du VPH, mesurée avec test PCR en nombre de copies/cellule au 1er et 3ième trimestre de grossesse, a été analysée en continue et dichotomisée à l’aide de différents seuils (0,5, 1,0 et 2,0). La charge virale (en continue) au 1er trimestre de grossesse a été associée à l’accouchement prématuré avec un OR ajusté (aOR) de 1,13 [IC 95% 1,03-1,25]. Le aOR pour la charge virale catégorisée avec seuil de 1,0 copie/cellule au 1er trimestre était de 15,03 [IC 95 % 1,75- 129,26]. Les analyses avec des seuils différents et au 3ième trimestre de grossesse ont données des résultats similaires quoique les ORs n’étaient pas toujours statistiquement significatifs. Nos résultats suggèrent une forte association entre la charge virale du VPH-16 et l’accouchement prématuré. Cette étude contribue à une meilleure compréhension des mécanismes tout en supportant la causalité. / Preterm birth has recently been associated with the persistence of human papillomavirus 16 (HPV-16) during pregnancy. However, it remains difficult to determine whether this association is causal and to explain its potential biological mechanisms. To better characterize this association, we investigated the association between HPV-16 viral load during pregnancy and preterm birth. Data from 48 pregnant women positive with HPV-16 infection from the HERITAGE cohort were analyzed using a logistic regression model, where confounders were adjusted with propensity scores and inverse probability treatment weighting. HPV viral load, measured with a PCR test as copy numbers/cell during the 1st and 3rd trimester of pregnancy was analyzed continuously, and categorized using different cutoffs (0.5, 1.0 and 2.0). Continuous viral load at 1st trimester of pregnancy was associated with preterm birth with an adjusted OR (aOR) of 1.13 [95% CI: 1.03-1.25]. The aOR viral load categorized with cutoff 1 copy/cell at 1st trimester was 15.03 [1.75-129.26]. Analyses with different cutoffs in 3rd trimester of pregnancy gave similar results although the ORs were not always statistically significant. Our results suggest a strong association between HPV-16 viral load and preterm birth. This study contributes to a better understanding of the mechanisms and provides an additional argument on causality.

This thesis focuses on the effects of the human papillomavirus (HPV) in tonsillar cancer, mobile tongue cancer, and recurrent respiratory papillomatosis (RRP). The purpose was to characterize patients with RRP in northern Sweden in order to identify more care-intensive RRP patients and to describe the voice and quality of life aspects that follow RRP. Further aims were to confirm the expected increase of HPV-positive tonsillar cancer cases in northern Sweden, and to study the correlation between HPV, its surrogate marker p16 and HPV receptor syndecan-1 in both tonsillar cancer and mobile tongue cancer. A total of 27 consecutive patients with RRP were evaluated at 3 months postoperatively using the voice handicap index (VHI) and SF-36 questionnaires to assess the impact on life and voice in a RRP population. The values were compared to normative data. This report was further extended by examining consecutive data from 21 new patients in order to characterize RRP patients in northern Sweden. In order to study HPV DNA in tonsillar (n= 65) and mobile tongue cancer (n=109), HPV DNA was extracted from paraffin-embedded biopsies and detected by polymerase chain reaction using general primers Gp5+/6+ and CpI/IIG. Expression of HPV surrogate marker p16 and the HPV receptor syndecan-1 was analysed by immunohistochemistry. Patients that underwent more than one RRP surgery per year were younger than those treated less frequently and they had significantly impaired voice quality as compared to normal subjects. Females, patients with frequent surgical treatment sessions, and patients with the high-risk HPV subtypes scored significantly lower in several domains of the quality of life assessment as compared with normal subjects. Forty-eight RRP patients had a median age of 44.5 years; 71% were men and 29% females, preferentially infected with HPV6. Patients with high surgical treatment frequency/year showed more widespread RRP in the larynx compared to the patients treated less frequently. A total of 214 tonsillar cancer cases were identified. The vast majority were men. They had a median age of 58 years at diagnosis and expressed HPV as well as p16. The incidence of tonsillar cancer revealed a 2,7-fold increase in men between the years 1990 and 2013. The study demonstrates a strong association between p16 and HPV infection in tonsillar malignancies. These findings are in contrast to the mobile tongue cancer cases, where no evidence of HPV DNA could be detected although one-third showed p16 staining. This demonstrated a poor correlation between HPV and p16 in mobile tongue cancer. There was no difference in the expression of the primary HPV receptor, syndecan-1, between tonsillar and mobile tongue cancer. In conclusion, the frequency of RRP operations, age at onset, gender and subtype of the HPV may be used as factors to predict voice disability. RRP patients with high surgical treatment frequency were significantly younger and had a more widespread laryngeal disease compared to the low-frequency treated group. This study confirms the existence of a clinical RRP group, not primarily related to HPV subtype, but to a more care-intensive RRP population. Our findings identify a 2,7-fold increase in the incidence of tonsillar cancer, HPV and p16 in men between 1990-2013. We can use p16 to detect HPV in tonsillar cancer but not in tongue cancer. The introduction of vaccination against HPV may have a role in the prevention of specific HPV-subtype positive head and neck malignancies and recurrent respiratory papillomatosis since the current vaccine protects against HPV6, 11, 16, 18, 31, 33, 45, 52 and 58. Males will definitely benefit indirectly from vaccination of females, though males will still remain at risk of cancers associated with HPV. This highlights the need for sex-neutral vaccination strategy. Our intention is that this thesis will provide scientific data to support a gender-neutral vaccination and to develop simple tools to detect HPV in tonsillar cancer. / Syftet med avhandlingen är att beskriva effekterna av humant papillomvirus (HPV) vid cancer i halsmandlarna, cancer i tungan och vid luftvägspapillom. Totalt 27 patienter med luftvägspapillom (RRP) under åren 2004-2012 utvärderades 3 månader efter operationen med röst handikapp index (VHI) och livskvalitetformuläret SF-36. Resultaten jämfördes med normal data. Studiematerialet utökades med 21 patienter till totalt 48 RRP patienter i syfte att karakterisera patientgruppen i norra Sverige. För att studera HPV-DNA i tonsillcancer (n = 65) och i cancer i mobil del av tungan (n = 109) extraherades HPV-DNA från paraffininbäddade provbitar som sedan analyserades med PCR teknik och GP5 + / 6 + och CPI/IIG primer. Uttryck av surrogatmarkör p16 och HPV-receptorn syndekan -1 analyserades med immunhistokemi. RRP patienter hade en medianålder på 44,5 år; 71% var män och 29% kvinnor, företrädesvis infekterade med HPV6. Patienter som opererades mer än en gång per år var yngre än de som behandlats mindre ofta och hade en statistiskt sämre röstkvalitet än friska kontroller. Kvinnor, patienter med täta kirurgiska behandlingsintervall och högrisk-HPV hade signifikant sämre livskvalitet jämfört med friska kontroller. Patienter med hög kirurgisk behandlingsfrekvens per år var signifikant yngre och hade mer utbredd RRP sjukdom i luftstrupen, jämfört med gruppen med låg behandlingsfrekvens. Sammanlagt, 214 fall av halsmandelscancer identifierades i norra Sverige under åren 1990-2013; majoriteten var män, med en medianålder på 58 år och positiva för både HPV och p16. Andelen halsmandelscancer fall ökade med 2,7 gånger bland männen på 23 år. Vi fann ett starkt samband mellan uttryck av p16 och HPV infektion i halsmandelscancer men inte i HPV-negativ, delvis p16-positiv (33%) mobil tungcancer. Det fanns ingen skillnad i uttrycket av den primära HPV-receptorn, syndekan -1, jämförande tung-, och halsmandelscancer. Antalet RRP operationer, ålder vid insjuknandet, kön och genetisk variant av HPV kan användas som indikatorer för att förutsäga grad av röststörning. RRP patienter med hög kirurgisk behandlingsfrekvens var signifikant yngre och hade en mer utbredd luftvägssjukdom jämfört med RRP patienter som behandlas mindre ofta. Vi har identifierat en undergrupp av RRP patienter som inte primärt karakteriseras efter HPV virusets genetik utan av ett mer vårdintensivt förlopp. Den aktuella avhandlingen har identifierat en 2,7-faldig ökning av antalet halsmandelscancer hos män och ett starkt samband mellan p16 och HPV infektion i halsmandlar men inte i HPV-negativ tungcancer som inte korrelerar till p16 uttryck. Vi kan använda p16 för att påvisa HPV i tonsillcancer men inte i cancer i mobil tunga. Idag ingår HPV vaccination i det allmänna vaccinationsprogrammet för flickor. Vi förväntar oss en tydlig profylaktisk effekt avseende insjuknande i HPV-relaterad huvud- och hals cancer samt luftvägspapillom eftersom vaccinet skyddar mot HPV bl.a. 6, 11, 16 och 18. Män kommer definitivt att gynnas indirekt genom vaccination av kvinnor men kommer att ha fortsatt högre risk än kvinnor att insjukna i HPV relaterad cancer vilket understryker behovet av könsneutral vaccination. Vår avsikt med avhandlingen är att ge vetenskapligt stöd för könsneutralt vaccination och enkla metoder att påvisa halsmandelscancer.

Cervical cancer is a major cause of death in women and is strongly associated with infection by human papillomavirus (HPV). Integration of HPV is thought to form a key step in the formation of cancer, and is thought to involve the upregulation of HPV E6 and E7 due to the loss of E2 transcriptional control. Leptomycin B (LMB), a nuclear export inhibitor, has previously been shown to induce apoptosis in HPV-containing cancer cell lines and HPV 16 E7 or E6/E7 transduced primary keratinocytes, but not in normal cells. This thesis shows that LMB can induce apoptosis and a reduction in the colony survival of derivatives of the W12 cell line that contain HPV 16 in either episomal or integrated form. The HPV genome status, including variations in viral integration type, appears to influence the cumulative and temporal pattern of LMB-induced apoptosis. The effects of LMB were also apparent in cells grown in organotypic raft culture, with differences in behaviour again apparent between cells containing episomal and integrated HPV. As previously noted, treatment with LMB was associated with increased expression of the cell regulators p53 and p21; however, the induction of apoptosis was not dependent upon transcriptionally active p53. It is therefore likely that induction and mediation of LMB-induced apoptosis occurs via alternative, currently unidentified, pathways. These findings suggest that LMB can induce apoptosis in keratinocytes containing HPV 16 in either episomal or integrated form, with genome status and potentially lesion grade likely to influence the response of HPV-associated anogenital lesions to LMB treatment.

O HPV é transmitido principalmente pelo contato sexual e as infecções causadas por tipos virais oncogênicos estão etiologicamente associadas com o desenvolvimento de câncer de colo de útero, vulva e ânus nas mulheres, câncer de pênis e ânus nos homens, e câncer de cabeça e pescoço em ambos os sexos. Além disso, as verrugas genitais e a rara, mas séria, papilomatose respiratória estão etiologicamente associadas aos HPVs de baixo risco 6 e 11. Ademais, os HPV-16 e 6 estão entre os tipos mais frequentemente detectados em homens, independentemente da origem da amostra estudada, ressaltando a importância epidemiológica do HPV-6. Até o momento, estudos de associação entre variantes moleculares de HPV e o desenvolvimento das doenças associadas foram realizados para os HPVs de alto-risco oncogênico, como os HPV-16 e -18. Em relação à prevalência dos HPVs de baixorisco oncogênico e as implicações da heterogeneidade viral, os dados existentes até o momento são escassos. Pelo exposto, este projeto tem por objetivo: (1) Determinar a prevalência das diferentes variantes moleculares de HPV-6 em esfregaços genitais e lesões genitais externas (LGE), especificamente em verrugas genitais (VGs), entre os participantes do estudo prospectivo multinacional da Infecção por HPV em homens (estudo HIM); (2) Verificar a associação entre a infecção por diferentes variantes moleculares de HPV-6 e o risco de desenvolvimento de LGE nos participantes do estudo HIM. Para atingir os objetivos propostos foram utilizados esfregaços genitais e amostras de verruga genital dos participantes HPV-6 positivos do estudo HIM. Nestas amostras, as variantes de HPV-6 foram caracterizadas através da amplificação por PCR e sequenciamento de um fragmento do gene L2. Isto permitiu classificar as amostras em todas as linhagens (A, B) e sub-linhagens (B1, B2, B3, B4, B5) de HPV-6 descritas. Neste estudo, as variantes da sub-linhagem B3 foram as mais prevalentes. A distribuição das variantes de HPV-6 diferiu entre os países e entre casos e controles. A prevalência das variantes B1 de HPV-6 estava aumentada em VGs e esfregaços genitais de casos em comparação aos controles. Diferenças entre a detecção de variantes B1 e B3 nas VG e no esfregaço genital precedente à lesão foram observadas. Foi encontrada uma associação significativa entre a detecção de variantes da sub-linhagem B1 de HPV-6 e o desenvolvimento de VGs. Em conclusão, variantes B1 de HPV-6 são mais prevalentes em esfregaços genitais normais que precedem o desenvolvimento de VGs. Ademais as variantes B1 conferem risco aumentado para o desenvolvimento de VGs. Estudos futuros são necessários para compreender o possível envolvimento aumentado de variantes B1 de HPV-6 na progressão para lesões clinicamente relevantes / HPV is primarily transmitted through sexual contact and infections caused by oncogenic viral types are etiologically associated with the development of cervical, vulvar and anal cancer, in women, penile and anal cancer in men, and head and neck cancer in both sexes. Moreover, genital warts and the rare, but serious, respiratory papillomatosis are etiologically associated with low-risk HPV types -6 and -11. Additionally, data obtained from different studies show that HPV types -16 and -6 are among the most frequently detected types in men, independently of the origin of the samples studied, underscoring the epidemiological relevance of HPV-6. To date, studies focusing on the association between HPV molecular variants and disease onset have been conducted on high-risk types such as -16 and -18. Regarding the prevalence of low-risk HPVs and the implications of their viral heterogeneity, date is still scarce. In light of these facts, the objectives of this project are to: (1) Determine the prevalence of HPV-6 molecular variants in genital swabs and external genital lesions (EGL), specifically genital warts (GW), among participants of the prospective and multinational HPV infection in men study (HIM study); (2) To verify the association between HPV-6 molecular variants infection and the risk of developing EGL among HIM study participants. To achieve the proposed objectives, genital swabs and genital wart samples from HPV-6 positive HIM study participants were used. In these samples, HPV-6 variants were characterized by PCR amplification followed by sequencing of an L2 gene fragment. This allowed for the classification of the samples into all described HPV-6 lineages (A, B) and sub-lineages (B1, B2, B3, B4, B5). In this study, variants belonging to B3 sub-lineage were the most prevalent. HPV-6 variants distribution differed between countries and between cases and controls. HPV-6 B1 variants prevalence was increased in GWs and genital swabs of cases compared to controls. Differences among B1 and B3 variants detection in GW and the preceding genital swab were observed. A significant association of HPV-6 B1 variants detection with GW development was found. In conclusion, HPV-6 B1 variants are more prevalent in normal genital swabs that precede GW development. Additionally, B1 variants confer an increased risk for GW development. Further research is needed to understand the possible increased involvement of B1 variants in the progression to clinically relevant lesions

Introdução. O Papilomavírus humano (HPV), particularmente o tipo 16, têm sido associado com risco e prognóstico de tumores de cabeça e pescoço. Contudo, o papel do DNA do HPV e resposta sorológica na sobrevida neste grupo de pacientes ainda não está claro. Objetivos. Avaliar o efeito do HPV (resposta sorológica e detecção do DNA no tecido tumoral) na sobrevida de pacientes com carcinoma epidermóide de cabeça e pescoço, considerando-se as distintas localizações anatômicas (cavidade oral, orofaringe, hipofaringe e laringe). Material e métodos. Coorte de 1.475 pacientes com carcinoma epidermóide de cabeça e pescoço, oriundos de dois estudos multicêntricos, diagnosticados entre novembro de 1998 e dezembro de 2008 e acompanhados até 30 de junho de 2009. Detecção de DNA do HPV no tecido tumoral foi feita pela técnica de PCR (Polymerase Chain Reaction) em tecido fresco e material parafinado. Resposta sorológica às proteínas do HPV foi determinada pela técnica Multiplex Luminex. Sobrevida global e específica pela doença foram calculadas pelo método atuarial (tábuas de vida). Curvas de sobrevida de Kaplan-Meier e teste Log-rank para comparação de curvas de sobrevida foram calculados. Hazard ratio (HR) do efeito da infecção pelo HPV nos tumores de cabeça e pescoço e respectivo intervalo com 95 por cento de confiança (IC95 por cento ) foram calculados via modelo de regressão de Cox ajustado pelas variáveis: estudo de origem dos casos, sexo, idade, educação, consumo de tabaco e de álcool, estadiamento do tumor e tratamento, assim como hábitos sexuais para a subcoorte com esta informação. Resultados. Prevalência de DNA do HPV 16 no tecido tumoral foi de 6,7 por cento nos casos recentes (2003-2008) comparado com 1 por cento nos casos iniciais (1998-2002) para a subcoorte de São Paulo. Aumento da soropositividade para HPV 16 E7 nos casos do estudo mais recente (2003-2008) comparado com os casos do estudo inicial (1998-2002) resultou estatisticamente significante. Foi observada pobre concordância entre os resultados de sorologia e DNA do HPV. Pacientes mais jovens (50 anos), que não fumavam e com tumores de orofaringe apresentaram risco aumentado na resposta sorológica à proteína E6 do HPV 16. Prática de sexo oral associou-se com resposta sorológica conjunta às proteínas E6 e E7 do HPV 16. Pacientes soropositivos para HPV 16 E6 apresentaram maior sobrevida global (HR=0,62; IC95 por cento =0,44-0,87), adicionalmente observou-se que a resposta sorológica conjunta às proteínas E6 e E7 do HPV incrementou a sobrevida dos pacientes com tumores de cabeça e pescoço (HR=0,34; IC95 por cento 0,17-0,70) e de orofaringe (HR=0,17; IC95 por cento 0,05-0,59). Conclusões. A prevalência da infecção pelo HPV entre pacientes com tumores de cabeça e pescoço aumentou no período estudado. O estudo sugere que resposta sorológica conjunta às proteínas E6 e E7 do HPV 16 pode estar associada com maior sobrevida global nos tumores de cabeça e pescoço, especificamente nos pacientes com tumores de orofaringe / Introduction. Human papillomavirus (HPV), especially type 16, had been associated with risk and prognostic of head and neck cancer. However, the role of HPV DNA and serological response in survival of patients with head and neck cancer is not yet clear. Objectives. Evaluate the effect of HPV (serological response and HPV DNA tumor status) in survival of patients with squamous cell carcinoma of head and neck (SCCHN), according to anatomical sites (oral cavity, oropharynx, hypopharynx and larynx). Material and methods. Cohort of 1,475 patients with SCCHN, from two multicentre studies diagnosed between November 1998 and December 2008 and followed-up until 30 of June 2009. HPV DNA detection was evaluated by PCR (Polymerase Chain Reaction) in fresh tissue and paraffin blocks. Antibodies to HPV in the serum were determinated by Multiplex Luminex technique. Overall and disease specific survival were calculated by actuarial method. Kaplan-Meier survival curves and Log-rank test in order to compare survival curves were calculated. Hazard ratio (HR) and 95 per cent of interval confidence (95 per cent IC) for Cox model regression were used to evaluate the effect of HPV infection in SCCHN, adjusted by variables: study group, sociodemographics, tobacco and alcohol consumption, tumor clinical stage and treatment, and also sexual habits in the subcohort with available information. Results. HPV DNA 16 tumoral status prevalence was 6.7 per cent in recent cases (2003-2008) compared to 1 per cent in old cases (1998-2002), only for subcohort of São Paulo. Seropositivity to HPV 16 E7 increased in the late cases (2003-2008) compared to old cases (1998-2002) and was statistically significant. Poor concordance was showed between DNA HPV and serological response to HPV. Younger patients (50 years old), no smokers and with oropharyngeal tumors showed increased risk to HPV 16 E6 serological response. Oral sex was associated with HPV 16 E6 and E7 simultaneously. Seropositivity to HPV 16 E6 had longer overall survival (HR=0.62; 95 per cent CI=0.440.87), additionally HPV 16 E6 and E7 serological response increased the survival of head and neck cancers (HR=0.34; 95 per cent CI 0.17-0.70) and oropharyngeal cancer (HR=0.17; 95 per cent CI 0.05-0.59) Conclusions. Prevalence of HPV infection in patients with SCCHN had increased in the study period. The study suggests that HPV 16 E6 serological response could be associated with increased overall survival in patients with SCCHN tumors and specifically with oropharyngeal cancer