Lalle Hammarstedt

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.

Human papillomavirus as a risk factor for the increase in incidence of tonsillar cancer.

Smoking and alcohol are well‐known etiological factors in tonsillar cancer. However, as in cervical cancer, human papillomavirus (HPV) is currently found in a sizable proportion of tonsillar cancer. Recent reports from the U.S. and Finland show an increase in the incidence of tonsillar cancer, without a parallel rise in smoking and alcohol consumption. This study investigates whether the incidence of tonsillar cancer has also changed in Sweden and whether a possible explanation of the increase is a higher proportion of HPV‐positive tonsillar cancer. The incidence of tonsillar cancer between 1970 and 2002 in the Stockholm area was obtained from the Swedish Cancer Registry. In parallel, 203 pretreatment paraffin‐embedded tonsillar cancer biopsies taken during 1970–2002 from patients in the Stockholm area were tested for presence of HPV DNA by PCR. The incidence of tonsillar cancer increased 2.8‐fold (2.6 in men and 3.5 in women) from 1970 to 2002. During the same period, a significant increase in the proportion of HPV‐positive tonsillar cancer cases was observed, as it increased 2.9‐fold (p < 0.001). The distribution of HPV‐positive cases was 7/30 (23.3%) in the 1970s, 12/42 (29%) in the 1980s, 48/84 (57%) in the 1990s and 32/47 (68%) during 2000–2002. We have demonstrated a highly significant and parallel increase both in the incidence of tonsillar cancer and the proportion of HPV‐positive tumors. Hence, HPV may play an important role for the increased incidence of tonsillar cancer. This should definitely influence future preventive strategies as well as treatment for this type of 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.

Human papillomavirus is a favourable prognostic factor in tonsillar cancer and its oncogenic role is supported by the expression of E6 and E7

From 1970 to 2002 in the Stockholm area, we revealed a parallel three‐fold increase in the incidence of tonsillar cancer and the proportion of human papillomavirus (HPV) positive tonsillar cancer cases, indicating a possible role of HPV infection in this disease. We have now examined whether HPV and viral load in pre‐treatment tonsillar cancer biopsies correlates to disease prognosis, and whether the presence of HPV‐16 E6 and E7 mRNA could be ascertained. The presence of HPV‐16, but not viral load, in tonsillar cancer was shown to be a favourable prognostic factor for clinical outcome. Moreover, E6 and/or E7 were expressed in almost all assessable HPV‐16 positive cases, supporting an oncogenic role of HPV‐16 in tonsillar cancer.

The incidence of tonsillar cancer in Sweden is increasing

Conclusions: The incidence of tonsillar cancer in Sweden is increasing, particularly among men. Risk factors other than smoking may have contributed to the observed secular trend in men. In women, however, smoking can be a part of the explanation. Further studies to look at changes in other environmental factors, such as human papilloma virus (HPV) infection, are clearly warranted. Objectives: Head and neck cancer is related to smoking habits and smoking has decreased substantially during the last 30 years in Sweden. However, there is suspicion that the incidence of tonsillar cancer has increased in the last 30 years as it has in the USA and Finland, in spite of reduced prevalence of known risk factors. The time trends of oral and oropharygeal cancer have been studied in Sweden, but not tonsillar cancer specifically. Subjects and methods: We used the Swedish Cancer Registry to assess the secular trend of incidence of tonsillar cancer in Sweden since 1960. For comparison we investigated the incidence of other oral cancers and lung cancer, which are also smoking-related. The prevalence of smoking was investigated for reference. Age-standardized incidence rates were calculated and linear regression was used to evaluate secular trends. Results: The incidence of tonsillar cancer increased by 2.6% per year in men and 1.1% in women. No similar increase was seen in the other oral cancers. For lung cancer there was a decrease in the incidence in men, but in women the incidence is still increasing.

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.

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.

Differential survival trends for patients with tonsillar, base of tongue and tongue cancer in Sweden

Tonsillar, base of tongue and tongue cancer have similar anatomical and histopathological appearances but present differences in prognosis. Human papillomavirus (HPV) is a known risk factor for tonsillar and base of tongue cancer, and a survival benefit has been shown for these tumors; however, HPV prevalence in tongue cancer is low. Tonsillar, base of tongue and tongue cancer patients registered in the Swedish Cancer Registry between 1960 and 2004 were followed from the date of cancer diagnosis until death, emigration out of Sweden, or the end of a follow-up (5 years since cancer diagnosis), whichever occurred first. The relative survival rate was computed as the ratio of the observed to the expected survival rate, in which the latter was inferred from the survival of the entire Swedish population in the same age, sex and calendar year stratum. The relative survival rate has improved significantly over time for patients with tonsillar and base of tongue cancer although delineated by different patterns. However, the relative survival rate in tongue cancer patients exhibited only a very modest improvement during the same time period. Contrary to the overall improved survival for patients with tonsillar and base of tongue cancer, the patients with tongue cancer show a very modest improvement in Sweden since 1960. Further studies are warranted to elucidate more effective treatment options for tongue cancer patients.

Presence of human papillomaviruses and p16 expression in hypopharyngeal cancer

Patients with hypopharyngeal cancer have a 5‐year survival of only 15% to 30%. Human papillomavirus (HPV) is a risk factor and a favorable prognostic factor for oropharyngeal carcinoma and p16 has been suggested as a surrogate marker for HPV‐induced cancer. However, few studies have been performed on HPV and p16 in hypopharyngeal cancer.