Linda Marklund

Incidence of human papillomavirus (HPV) positive tonsillar carcinoma in Stockholm, Sweden: An epidemic of viral-induced carcinoma?

In the county of Stockholm, between 1970 and 2002, we have previously reported a 3‐fold parallel increase in the incidence of tonsillar squamous cell carcinoma (SCC) and the proportion of human papillomavirus (HPV) positive tonsillar SCC. Here, we have followed the above parameters in all patients (n = 120) diagnosed with tonsillar SCC during 2003–2007 in the same area, and also in correlation to our previous data. Ninety‐eight pretreatment biopsies were available and presence of HPV DNA and HPV‐16 E6 and E7 RNA were tested by polymerase chain reaction (PCR) and RT‐PCR. Incidence data were obtained from the Swedish Cancer Registry. Data reported from 1970 to 2002 were also obtained for comparison. HPV DNA was present in 83 of 98 (85%) of the tonsillar SCC biopsies from 2003 to 2007 and 77 of these were HPV‐16 positive. HPV‐16 E6 and E7 RNA were found in 98% of 52 analyzed HPV‐16 positive cases. The proportion of HPV‐positive cancers had significantly increased both from 1970 to 2007 (p < 0.0001) as well from 2000 to 2007 (p < 0.01), with 68% (95% confidence interval (CI), 53–81) 2000–2002; 77% (95% CI, 63–87) 2003–2005; and 93% (95% CI, 82–99) 2006–2007. The incidence rate of HPV‐positive tumors almost doubled each decade between 1970 and 2007, in parallel with a decline of HPV‐negative tumors. In conclusion, the incidence of HPV‐positive cancers is still increasing in the County of Stockholm, suggesting an epidemic of a virus‐induced carcinoma, with soon practically all tonsillar SCC being HPV positive, as in cervical cancer.

The role of human papillomavirus in the increased incidence of base of tongue cancer

Numerous reports have shown that the incidence for oropharyngeal cancer is increasing and that human papillomavirus (HPV) is a risk factor. However, few studies have investigated the specific subsites of the oropharynx. Following our previous research on tonsillar cancer, we assessed the increase in the incidence of base of tongue cancer and the prevalence of HPV in this disease. Between 1998 and 2007, 109 patients were diagnosed for base of tongue cancer in Stockholm county. Ninety‐five paraffin‐embedded diagnostic tumor biopsies from patients were obtained and tested for HPV, both by general HPV PCR and HPV‐16/HPV‐33 type‐specific PCR. Expression of HPV‐16 RNA was analyzed to confirm E6 and/or E7 expression. Incidence data were obtained from the Swedish Cancer Registry. An overall increase in the incidence of base of tongue cancer from 0.15/100,000 person‐years during 1970–1974 to 0.47/100,000 person‐years during 2005–2007 was found in Sweden. The prevalence of HPV in base of tongue cancer in Stockholm county increased from 58% during 1998–2001 to 84% during 2004–2007 (p < 0.05). In the HPV‐positive tumors, HPV‐16 dominated (86%) but interestingly, HPV33 was detected in as many as 10%. E6 and/or E7 RNA were found in 85% of the samples tested. The incidence of base of tongue cancer, as well as the proportion of HPV‐positive tumors, has increased in Sweden during the study period, suggesting that HPV may contribute to this increase.

Tumor Infiltrating CD8+ and Foxp3+ Lymphocytes Correlate to Clinical Outcome and Human Papillomavirus (HPV) Status in Tonsillar Cancer

Background Human papillomavirus (HPV) is a causative factor for tonsillar squamous cell carcinoma (TSCC) and patients with HPV positive (HPV+) TSCC have a better clinical outcome than those with HPV negative (HPV−) TSCC. However, since not all patients with HPV+TSCC respond to treatment, additional biomarkers are needed together with HPV status to better predict response to therapy and to individualize treatment. For this purpose, we examined whether the number of tumor infiltrating cytotoxic and regulatory T-cells in TSCC correlated to HPV status and to clinical outcome. Methods Formalin fixed paraffin embedded TSCC, previously analysed for HPV DNA, derived from 83 patients, were divided into four groups depending on the HPV status of the tumor and clinical outcome. Tumors were stained by immunohistochemistry and evaluated for the number of infiltrating cytotoxic (CD8+) and regulatory (Foxp3+) T-cells. Results A high CD8+ T-cell infiltration was significantly positively correlated to a good clinical outcome in both patients with HPV+ and HPV- TSCC patients. Similarly, a high CD8+/Foxp3+ TIL ratio was correlated to a 3-year disease free survival. Furthermore, HPV+TSCC had in comparison to HPV−TSCC, higher numbers of infiltrating CD8+ and Foxp3+ T-cells. Conclusions In conclusion, a positive correlation between a high number of infiltrating CD8+ cells and clinical outcome indicates that CD8+ cells may contribute to a beneficial clinical outcome in TSCC patients, and may potentially serve as a biomarker. Likewise, the CD8+/Foxp3+cell ratio can potentially be used for the same purpose.

Human papillomavirus and survival in patients with base of tongue cancer.

The incidence of base of tongue cancer is increasing in Sweden and the proportion of human papillomavirus (HPV) positive cancer has increased in Stockholm, Sweden. Between 2006 and 2007, 84% of base of tongue cancer cases in Stockholm were HPV‐positive. The objective of this study was to assess the impact of HPV status on prognosis for base of tongue cancer patients. One‐hundred and nine patients were diagnosed with base of tongue cancer between 1998 and 2007 in Stockholm County and 95 paraffin‐embedded diagnostic tumor biopsies were obtained and tested for HPV by PCR. Eighty‐seven patients had available biopsies, were treated with intention to cure and could be included in the survival analysis. Age, sex, TNM‐stage, stage, treatment and survival were recorded from patient charts. Kaplan–Meier curves were used to present survival data. In multivariable analyses, a Cox proportional hazards model was used to adjust for covariates. In total 68 (78%) tumor biopsies from the 87 included patients were HPV DNA positive. Kaplan–Meier estimates showed that the overall survival for patients with HPV‐positive cancer was significantly better (p = 0.0004), (log‐rank test) than that of patients with HPV‐negative cancer. Patients with HPV‐positive tumors also had significantly better disease‐free survival (p = 0.0008), (log‐rank test) than those with HPV‐negative tumors. These results further strengthen the option to consider HPV‐status when planning prospective studies on treatment for base of tongue cancer.

CD8+ and CD4+ tumour infiltrating lymphocytes in relation to human papillomavirus status and clinical outcome in tonsillar and base of tongue squamous cell carcinoma.

Patients with human papillomavirus (HPV) positive tonsillar and base of tongue squamous cell carcinoma (TSCC and BOTSCC, respectively) have a better clinical outcome than those with HPV negative tumours, irrespective of treatment. However, to better individualise treatment, additional biomarkers are needed together with HPV status. In a pilot study, we showed that high numbers of CD8(+) tumour infiltrating lymphocytes (TILs) in HPVDNA+ p16(INK4a+) TSCC indicated a better outcome. Here this study was extended. Totally 203 TSCC and 77 BOTSCC formalin fixed paraffin embedded tumour biopsies, earlier tested for HPV DNA (79% HPVDNA+) and p16(INK4a) from patients treated with curative intention, were analysed for CD8(+) and CD4(+) TILs by immunohistochemistry. Data obtained for 275 patients were correlated to HPVDNA and p16(INK4a) status, overall survival (OS) and disease free survival (DFS). In both HPVDNA+ and HPVDNA+ p16(INK4a+) tumours higher CD8(+) TIL counts correlated to a better 3-year OS (logrank test, both p<0.001) and 3-year DFS (logrank test, p = 0.003 and p = 0.004 respectively) as compared to the lowest quartile in the groups. A similar pattern was observed when analysing TSCC alone, while for BOTSCC significance was obtained only for 3-year OS. In HPVDNA- tumours the trend was similar, but significance was obtained again only for 3-year OS. The number of CD4(+) TILs did not generally correlate to survival. In conclusion, in HPVDNA+ and/or HPVDNA+ p16(INK4a+) tumours high CD8(+) TIL counts indicated a better 3-year OS. This suggests that high CD8(+) TIL counts together with HPVDNA+ or HPVDNA+ p16(INK4a+) could be used when selecting patients for more individualised treatment.

Prevalence of human papillomavirus and survival in oropharyngeal cancer other than tonsil or base of tongue cancer.

Today, most oropharyngeal squamous cell carcinoma (OSCC) is human papillomavirus (HPV) positive and HPV alone or in combination with p16 is reported to be a favorable prognostic factor for OSCC. Patients with tumors at other OSCC sites (OOSCC) are often included in the same treatment and study protocols as patients with tonsillar‐ and base of tongue SCC, even though the prevalence and clinical significance of HPV infection in OOSCC is unknown. Since tonsillar and base of tongue SSC cover roughly 90% of all OSCC, there is an obvious risk that there may be a misinterpretation of the results for OOSCC. Herein, we therefore study the prevalence of HPV and p16 and their impact on survival in OOSCC. A total of 69 patients were included in the study, and 61 were included in the survival analysis. HPV and p16 were present in only 17% (12/69) and 25% (17/69) of the OOSCC cases, respectively, while the majority 69% (48/69) was both HPV and p16 negative. Neither HPV nor p16 had predictive value for clinical outcome in OOSCC in this study. In conclusion, the prevalence of HPV and/or p16 is much lower in OOSCC compared to earlier reports including all OSCC, or tonsillar‐ and base of tongue cancer alone and HPV and p16 had no impact on clinical outcome in OSCC in this study. Our data highlight the diversity of head neck cancer sub‐sites and the importance of taking OSCC sub‐sites in consideration in future clinical trials and treatment.

Impact of HPV in Oropharyngeal Cancer.

The incidence of oropharyngeal cancers has increased in the western world and Human Papilloma Virus (HPV) has been recognised as a risk factor in the last decades. During the same period the prevalence of HPV in oropharyngeal tumours has increased and HPV has been suggested responsible for the increase. The HPV-positive tumours are today recognized as a distinct subset of head and neck cancers with its own clinopathological and risk profile and have a significantly improved prognosis regardless of treatment strategy. This review summarizes current knowledge regarding human papillomavirus biology, oncogenic mechanisms, risk factors, and impact of treatment.

Incidence of human papillomavirus positive tonsillar and base of tongue carcinoma: A stabilisation of an epidemic of viral induced carcinoma?

AIM To investigate whether the rise during the past decades in the incidence of tonsillar and base of tongue squamous cell carcinoma (TSCC and BOTSCC) and the proportion of human papillomavirus (HPV) positive cancer has continued in Stockholm. PATIENTS AND METHODS Pre-treatment biopsies (n=252) available from 280 patients diagnosed with TSCC and BOTSCC during 2008-2012 in the County of Stockholm were tested for HPV DNA by a multiplex bead-based assay. Incidence records were acquired from the Swedish Cancer Registry. The data obtained were evaluated together with previous figures from 1970 to 2007. RESULTS HPV DNA was present in 186/252 (74%) of TSCC and BOTSCC biopsies obtained during 2008-2012 in Stockholm. In this region the age-standardised incidence, including the prevalence of HPV-positive and HPV-negative TSCC stabilised 2007-2012 compared to 2000-2006, while for BOTSCC throughout 1998-2012 the same parameters increased moderately (p<0.05, for all). In parallel, from 2000 to 2006 through 2007-2012 in Sweden, the age-standardised incidence of both TSCC and BOTSCC continued to rise (p=0.012 and p=0.001 respectively). CONCLUSION During 2000-2012 the age-standardised incidence and the proportion of HPV-positive TSCC have stabilised at a high level, while the proportion of HPV-negative cancer has remained at a low level in Stockholm, whereas for BOTSCC all parameters are increasing moderately. In contrast, in Sweden the incidence of both TSCC and BOTSCC is still increasing. We hypothesise that the HPV epidemic could be stabilising, first for TSCC, but so far not for BOTSCC, in e.g. some urban areas, while previous trends for both tumours persist at other geographic locations.

HLA Class I and II Expression in Oropharyngeal Squamous Cell Carcinoma in Relation to Tumor HPV Status and Clinical Outcome

HPV-DNA positive (HPVDNA+) oropharyngeal squamous cell carcinoma (OSCC) has better clinical outcome than HPV-DNA negative (HPVDNA-) OSCC. Current treatment may be unnecessarily extensive for most HPV+ OSCC, but before de-escalation, additional markers are needed together with HPV status to better predict treatment response. Here the influence of HLA class I/HLA class II expression was explored. Pre-treatment biopsies, from 439/484 OSCC patients diagnosed 2000-2009 and treated curatively, were analyzed for HLA I and II expression, p16INK4a and HPV DNA. Absent/weak as compared to high HLA class I intensity correlated to a very favorable disease-free survival (DFS), disease-specific survival (DSS) and overall survival (OS) in HPVDNA+ OSCC, both in univariate and multivariate analysis, while HLA class II had no impact. Notably, HPVDNA+ OSCC with absent/weak HLA class I responded equally well when treated with induction-chemo-radiotherapy (CRT) or radiotherapy (RT) alone. In patients with HPVDNA- OSCC, high HLA class I/class II expression correlated in general to a better clinical outcome. p16INK4a overexpression correlated to a better clinical outcome in HPVDNA+ OSCC. Absence of HLA class I intensity in HPVDNA+ OSCC suggests a very high survival independent of treatment and could possibly be used clinically to select patients for randomized trials de-escalating therapy.

Absent/weak CD44 intensity and positive human papillomavirus (HPV) status in oropharyngeal squamous cell carcinoma indicates a very high survival

Patients with human papillomavirus DNA positive (HPVDNA+) oropharyngeal squamous cell carcinoma (OSCC) have better clinical outcome than those with HPV DNA negative (HPVDNA−) OSCC upon intensive oncological treatment. All HPVDNA+ OSCC patients may not require intensive treatment, however, but before potentially deintensifying treatment, additional predictive markers are needed. Here, we examined HPV, p16INK4a, and CD44 in OSCC in correlation to clinical outcome. Pretreatment tumors from 290 OSCC patients, the majority not receiving chemotherapy, were analyzed for HPV DNA by Luminex and for p16INK4a and CD44 by immunohistochemistry. 225/290 (78%) tumors were HPVDNA+ and 211/290 (73%) overexpressed p16INK4a, which correlated to presence of HPV (P < 0.0001). Presence of HPV DNA, absent/weak CD44 intensity staining correlated to favorable 3‐year disease‐free survival (DFS) and overall survival (OS) by univariate and multivariate analysis, and likewise for p16INK4a by univariate analysis. Upon stratification for HPV, HPVDNA+ OSCC with absent/weak CD44 intensity presented the significantly best 3‐year DFS and OS, with >95% 3‐year DFS and OS. Furthermore, in HPVDNA+ OSCC, p16INK4a+ overexpression correlated to a favorable 3‐year OS. In conclusion, patients with HPVDNA+ and absent/weak CD44 intensity OSCC presented the best survival and this marker combination could possibly be used for selecting patients for tailored deintensified treatment in prospective clinical trials.