Mircea Romanitan

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.

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.

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.

Prevalence of Oral Human Papillomavirus Infection among Youth, Sweden

Human papillomavirus (HPV) causes cervical, head, and neck cancers. We studied 483 patients at a youth clinic in Stockholm, Sweden, and found oral HPV prevalence was 9.3% and significantly higher for female youth with than without cervical HPV infection (p = 0.043). Most oral HPV types matched the co-occurring cervical types.

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.

Human papillomavirus (HPV) 16 E6 variants in tonsillar cancer in comparison to those in cervical cancer in Stockholm, Sweden.

Background Human papillomavirus (HPV), especially HPV16, is associated with the development of both cervical and tonsillar cancer and intratype variants in the amino acid sequence of the HPV16 E6 oncoprotein have been demonstrated to be associated with viral persistence and cancer lesions. For this reason the presence of HPV16 E6 variants in tonsillar squamous cell carcinoma (TSCC) in cervical cancer (CC), as well as in cervical samples (CS), were explored. Methods HPV16 E6 was sequenced in 108 TSCC and 52 CC samples from patients diagnosed 2000–2008 in the County of Stockholm, and in 51 CS from young women attending a youth health center in Stockholm. Results The rare E6 variant R10G was relatively frequent (19%) in TSCC, absent in CC and infrequent (4%) in CS, while the well-known L83V variant was common in TSCC (40%), CC (31%), and CS (29%). The difference for R10G was significant between TSCC and CC (p = 0.0003), as well as between TSCC and CS (p = 0.009). The HPV16 European phylogenetic lineage and its derivatives dominated in all samples (>90%). Conclusion The relatively high frequency of the R10G variant in TSCC, as compared to what has been found in CC both in the present study as well as in several other studies in different countries, may indicate a difference between TSCC and CC with regard to tumor induction and development. Alternatively, there could be differences with regard to the oral and cervical prevalence of this variant that need to be explored further.

Studies on human papillomavirus (HPV) 16 E2, E5 and E7 mRNA in HPV-positive tonsillar and base of tongue cancer in relation to clinical outcome and immunological parameters

OBJECTIVES Three-year survival is 80% for human papillomavirus (HPV) positive tonsillar and base of tongue squamous cell carcinoma (TSCC and BOTSCC) and higher (95-100%) in patients with tumors without HLA class I expression, or with high CD8(+) tumor-infiltrating lymphocyte (TIL) counts. The former paradoxical, the latter expected, but it is known that E5 and E7 can downregulate HLA class I expression. Furthermore, upon HPV integration, E2, sometimes in combination with E5 is lost. Here, HPV16 E2, E5 and E7 mRNA was therefore examined in relation to HLA class I expression, TIL counts and survival. PATIENTS AND METHODS HPV16 DNA positive TSCC and BOTSCC biopsies, analyzed for HLA class I and CD8(+) TILs, of 133 patients, treated curatively between 2000 and 2011, were tested for HPV16 E2, E5 and E7 mRNA expression. Totally 127 samples could be evaluated and of these 117 patients, all with HPV16/E7-mRNA-positive tumors, were included in the final analysis. RESULTS Most tumors (92%) expressed E7 mRNA, and of these 64% also expressed E2 and E5 mRNA. Patients with tumors lacking E2 mRNA had worse 3-year relapse and progression free survival (p<0.01 and p<0.05), while presence of E5 had no impact on clinical outcome. Furthermore, HLA class I expression and TILs were not correlated to E5 or to E2 mRNA expression. CONCLUSION Lack of E2 but not E5 mRNA in HPV16 positive TSCC and BOTSCC was a negative prognostic marker. Presence of HPV16 E2, E5 and E7 mRNA expression was not correlated to HLA class I expression or CD8(+) TILs.

Human papillomavirus DNA and p16INK4a expression in hypopharyngeal cancer and in relation to clinical outcome, in Stockholm, Sweden

OBJECTIVES Hypopharyngeal cancer is a subset of head neck squamous cell carcinoma (HNSCC) with particularly poor prognosis. Human papillomavirus (HPV) is a risk factor for some HNSCC, and its presence is of prognostic value for certain subsites. However, its influence on survival in hypopharyngeal cancer has not been thoroughly investigated. Here we examine HPV DNA and p16(INK4a) (p16) overexpression in relation to clinical outcome. MATERIALS AND METHODS Hypopharyngeal tumour biopsies from 82 patients diagnosed 2008-2013 were examined for presence of HPV DNA by a bead-based multiplex assay and for p16 expression by immunohistochemistry, and the obtained data compared to that acquired previously from 109 patients diagnosed 2000-2007 at the same clinic. A survival analysis was then performed on 142 patients (from both studies) treated with curative intent and a 3-year follow-up time. RESULTS Of the tumour biopsies 3/82 (3.7%) were HPV16 DNA and p16 positive, while 12/82 (14.6%) were p16 positive, equivalent to that in the previous study. Overall 3-year survival was significantly more favourable for patients with HPV16 DNA and p16 positive tumours as compared to survival of the other patients (86% vs. 31%, p=0.0185). A similar but not statistically significant trend was found for disease specific survival. CONCLUSION HPV DNA and p16 positive hypopharyngeal cancer was rare and had not increased, but had a better clinical outcome as compared to other HPV-unrelated hypopharyngeal cancer. In addition, p16 overexpression was not a suitable surrogate marker for presence of HPV or for prediction of survival in this type of cancer.

MicroRNA-155, -185 and -193b as biomarkers in human papillomavirus positive and negative tonsillar and base of tongue squamous cell carcinoma

OBJECTIVE Three-year disease-free survival (DFS) is 80% for human papillomavirus (HPV) positive tonsillar and base of tongue cancer (TSCC/BOTSCC) treated with radiotherapy alone, and todays intensified therapy does not improve prognosis. More markers are therefore needed to more accurately identify patients with good prognosis or in need of alternative therapy. Here, microRNAs (miRs) 155, 185 and 193b were examined as potential prognostic markers in TSCC/BOTSCC. MATERIAL AND METHODS 168 TSCC/BOTSCC patients diagnosed 2000-2013, with known data on HPV-status, CD8+ tumour infiltrating lymphocytes, tumour staging and survival were examined for expression of miR-155, -185 and -193b using Real-Time PCR. Associations between miR expression and patient and tumour characteristics were analysed using univariate testing and multivariate regression. RESULTS Tumours compared to normal tonsils showed decreased miR-155 and increased miR-193b expression. miR-155 expression was associated with HPV-positivity, low T-stage, high CD8+ TIL counts and improved survival. miR-185 expression was associated with HPV-negativity and a tendency towards decreased survival, while miR-193b expression was associated with higher T-stage, male gender and lower CD8+ TIL counts, but not with outcome. Upon Cox regression, miR-185 was the only miR significantly associated with survival. Combining miR-155 and miR-185 to predict outcome in HPV+ patients yielded an area under curve (AUC) of 71%. CONCLUSION Increased miR-155 expression was found as a positive predictor of survival, with the effect mainly due to its association with high CD8+ TIL numbers, while miR-185 independently associated with decreased survival. Addition of these miRs to previously validated prognostic biomarkers could improve patient stratification accuracy.