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OBJECTIVES: Recurrent respiratory papillomatosis (RRP) is a relatively rare, chronic disease caused by Human Papilloma Virus (HPV) 6 and 11, and characterized by wart-like lesions in the airway affecting voice and respiratory function. The majority of HPV infections are asymptomatic and resolve spontaneously, however, some individuals are afflicted with persistent HPV infections. Failure to eliminate HPV 6 and 11 due to a defect immune responsiveness to these specific genotypes is proposed to play a major role in the development of RRP.

METHODS: We performed a phenotypic characterization of peripheral blood mononuclear cells (PBMC) collected from 16 RRP patients and 12 age-matched healthy controls, using immunoflow cytometry, and monoclonal antibodies against differentiation and activation markers. The cytokine mRNA profile of monocytes, T helper-, T cytotoxic-, and NK cells was assessed using RT-qPCR cytokine analysis, differentiating between Th1-, Th2-, Th3/regulatory-, and inflammatory immune responses.

RESULTS: We found a dominance of cytotoxic T cells, activated NK cells, and high numbers of stressed MIC A/B expressing lymphocytes. There was an overall suppression of cytokine mRNA production and an aberrant cytokine mRNA profile in the activated NK cells.

CONCLUSION: These findings demonstrate an immune dysregulation with inverted CD4(+) /CD8(+) ratio and aberrant cytokine mRNA production in RRP patients, compared to healthy controls.

Squamous cell carcinoma of the head and neck (SCCHN) comprises a large group of cancers in the oral cavity and nasopharyngeal area that typically arise in older males in association with alcohol/tobacco usage. Within the oral cavity, the mobile tongue is the most common site for tumour development. The incidence of tongue squamous cell carcinoma (TSCC) is increasing in younger people, which has been suggested to associate with a viral aetiology. Two common human oncogenic viruses, human papilloma virus (HPV) and Epstein-Barr virus (EBV) are known causes of certain types of SCCHN, namely the oropharynx and nasopharynx, respectively. EBV infects most adults worldwide through oral transmission and establishes a latent infection, with sporadic productive viral replication and release of virus in the oral cavity throughout life. In view of the prevalence of EBV in the oral cavity and recent data indicating that it infects tongue epithelial cells and establishes latency, we examined 98 cases of primary squamous cell carcinoma of the mobile tongue and 15 cases of tonsillar squamous cell carcinoma for the presence of EBV-encoded RNAs (EBERs), EBV DNA and an EBV-encoded protein, EBNA-1. A commercially available in situ hybridisation kit targeting EBER transcripts (EBER-ISH) showed a positive signal in the cytoplasm and/or nuclei of tumour cells in 43% of TSCCs. However, application of control probes and RNase A digestion using in-house developed EBER-ISH showed identical EBER staining patterns, indicating non-specific signals. PCR analysis of the BamH1 W repeat sequences did not identify EBV genomes in tumour samples. Immunohistochemistry for EBNA-1 was also negative. These data exclude EBV as a potential player in TSCC in both old and young patients and highlight the importance of appropriate controls for EBER-ISH in investigating EBV in human diseases.

Epigenetic modifications are essential regulators of biological processes. Decreased DNA methylation of OAS2 (2'-5'-Oligoadenylate Synthetase 2), encoding an antiviral protein, has been seen in psoriasis. To provide further insight into the epigenetic regulation of OAS2, we performed pyrosequencing to detect OAS2 DNA methylation status at 11 promoter and first exon located CpG sites in psoriasis (n = 12) and two common subtypes of squamous cell carcinoma (SCC) of the head and neck: tongue (n = 12) and tonsillar (n = 11). Compared to corresponding controls, a general hypomethylation was seen in psoriasis. In tongue and tonsillar SCC, hypomethylation was found at only two CpG sites, the same two sites that were least demethylated in psoriasis. Despite differences in the specific residues targeted for methylation/demethylation, OAS2 expression was upregulated in all conditions and correlations between methylation and expression were seen in psoriasis and tongue SCC. Distinctive methylation status at four successively located CpG sites within a genomic area of 63 bp reveals a delicately integrated epigenetic program and indicates that detailed analysis of individual CpGs provides additional information into the mechanisms of epigenetic regulation in specific disease states. Methylation analyses as clinical biomarkers need to be tailored according to disease-specific sites.

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.

Background: Tongue squamous cell carcinoma (TSCC) is increasing in incidence, especially among young patients and preferably females. Infection with human papilloma virus (HPV) has been suggested as a cause of SCC in the head and neck, and the proportion of oropharyngeal cancers caused by HPV has steadily increased. Methods: Samples from 109 patients with primary TSCC were analysed for the presence of HPV16 by in situ hybridisation and for expression of its surrogate marker p16 and the HPV receptor syndecan-1 by immunhistochemistry. Results: No evidence of HPV16 DNA was observed in the tumours, although one-third showed p16 staining. There was no difference in the expression of the primary HPV receptor, syndecan-1, between TSCC and a group of tonsil SCC. Conclusion: Whereas p16 is expressed in some TSCCs, HPV16 is undetectable, therefore, p16 cannot be used as a surrogate marker for high-risk HPV-infection in this tumour. Despite presence of the HPV-receptor syndecan-1 in TSCC, HPV prefers the tonsillar environment. Lack of p16 associates with worse prognosis primarily in patients aged <= 40 years with tongue SCC. The improved prognosis seen in p16-positive TSCC can be due to induction of a senescent phenotype or an inherent radiosensitivity due to the ability of p16 to inhibit homologous recombination repair.

The incidence rate of tonsillar cancer is increasing worldwide. The current study identifies a parallel increase in the incidence of tonsillar cancer, human papilloma virus (HPV) and p16 expression among a population from northern Sweden, a sparsely populated area, confirming the strong association between p16 and HPV infection in tonsillar tissue. Data from the Swedish Cancer Registry was assessed to identify cases of tonsillar cancer in the northern territorial area of Sweden. HPV DNA was extracted from paraffin embedded diagnostic biopsies and detected by polymerase chain reaction using general primers Gp5+/6+ and CpI/IIG. Expression of p16 was identified by immunochemistry. Patients were grouped into urban or rural residence categories. A total of 214 cases were identified, comprising 155 (72.4%) men and 59 (27.6%) women, and 65 of these patients, who presented between 2000 and 2012, were analyzed. The overall median age for the analyzed patients was 58 years; 48 (74%) were males (median age, 57.5 years) and 17 (26%) were females (median age, 65 years). Of the 65 specimens, 59 (91%) were positive for HPV, and 62 (95%) expressed p16. The incidence of tonsillar cancer in the cohort demonstrated a 2-fold increase between 1990 and 2013; specifically, a 2.7-fold increase was observed in men whilst the female group exhibited only a small increase. These findings demonstrate a strong association between p16 expression and HPV infection in tonsillar malignancies. The incidence of HPV-positive tonsillar cancer has increased in recent years, even in sparsely populated regions, as demonstrated in northern Sweden.

Conclusion: Recurrent respiratory papillomatosis (RRP) patients with high surgical treatment frequency (>= 1/year, HF) were significantly younger and had a more widespread laryngeal disease compared to a low frequency treated group (< 1 treatment/year, LF). This study confirms the existence of a clinical RRP group, not primarily related to HPV sub-type, but more care-intensive and in need of more vigilant follow-up. Objectives: RRP is associated with high morbidity due to its influence on breathing and voice. The purpose of this study was to characterize RRP patients in northern Sweden and investigate possible predictor factors affecting therapeutic needs. Method: Patients from the regional referral area (northern Sweden) were categorized for age, disease duration, juvenile or adult onset, profile of disease development, number of surgical sessions in relation to disease duration, laryngeal deposition of papilloma, gender, and HPV sub-types, in order to identify patients with increased need for frequent surgical treatment. Results: The median age of the RRP patients (n = 48) was 44.5 years; 34 (71%) were males and 14 (29%) females, most were infected with HPV 6. Patients with high surgical treatment frequency/year were significantly younger and showed more widespread papillomatous vegetation in the larynx, compared to the low frequency treated group.

Abstract Conclusion:

The frequency of operations, age at onset, gender and subtype of the human papilloma virus (HPV) may be used as factors to predict voice disability.

Objectives:

Patients with recurrent respiratory papillomatosis (RRP) are characterized by morbid consequences due to a lifelong repetitive influence on voice and breathing ability and the need for recurrent surgical treatments. The aim of the study was to measure the quality of voice and life using evaluated and validated questionnaires in a northern Sweden RRP population.

Methods:

A total of 27 consecutive patients with RRP (age 21-71 years, median 47 years) were evaluated 3 months postoperatively (CO2 laser treatment) using the voice handicap index (VHI) and SF-36 questionnaires to assess the impact on life and voice in an RRP population. The values were compared to historical normative data, VHI ≤ 20.

Results:

Patients that underwent more than one operation per year were younger (p = 0.028) than those treated less frequently. The mean VHItotal score in patients with RRP was 39.3, indicating a statistically significant impairment of voice quality (p < 0.001) as compared with normal subjects. Voice dysfunction was observed in 21 patients (78%). Significantly lower values than the normal population regarding the quality of life in patients with RRP were obtained in the domain of social functioning (p = 0.029). Females, patients with frequent surgical treatment sessions and patients with the high-risk HPV types scored significantly lower in several domains of the quality of life assessment as compared with normal subjects. The results should be interpreted with caution due to the limited number of subjects.

Laryngeal papilloma is associated with human papillomavirus (HPV) 6, 11, 16, 18 and 31. The variation in the frequency of surgical treatment between patients for the same subtypes of HPV is inconsistent and poorly understood. Comparisons of the female laryngeal papilloma group (n?=?8, median age 46 yrs) with the male (n= 18, median age 32 yrs) with respect to gender, age, time of disease, period of life for diagnosis, disease progression profile, frequency of surgery (CO2 laser) during time of disease, localisation of papilloma in the upper airway and HPV subtype did not reach significance. In contrast the comparison between the high frequency (Ž 1 treatment/yr, n?=?11, median age 31 yrs) and low frequency (<1 treatment/yr, n?=?15 median age 45 yrs) treatment groups with regard to the same parameters as the female-male comparison, showed a clear-cut higher median age in the low frequency group (P?=?0,01).

Ingen signifikant skillnad mellan könen noterades i denna studie av en grupp patienter i Norrland där man jämfört kvinnor (n = 8, medianålder 46 år) och män (n = 18, me­dianålder 32 år) med larynxpapillom med avseende på aktuell ­ålder, sjukdomsduration, dia­gnosperiod i livet, sjukdomsutveckling, operationstillfällen under sjukdomsperioden, ­papillomlokalisation i luft­vägar och HPV-typ. Brytpunkten för hög respek­tive låg behandlingsfrekvens förlades till medelvärdet 1 behandling/år. Vid jämförelse mellan patienter med hög (≥ 1 behandling/år, n = 11, medianålder 31 år) respektive låg behandlingsfrekvens (< 1 behandling/år, n = 15, medianålder 45 år) ­registrerades signifikant ­högre ålder i gruppen med låg behandlingsfrekvens (P = 0,01).