Tina Dalianis

Identification of a Third Human Polyomavirus

ABSTRACT We have previously reported on a system for large-scale molecular virus screening of clinical samples. As part of an effort to systematically search for unrecognized human pathogens, the technology was applied for virus screening of human respiratory tract samples. This resulted in the identification of a previously unknown polyomavirus provisionally named KI polyomavirus. The virus is phylogenetically related to other primate polyomaviruses in the early region of the genome but has very little homology (<30% amino acid identity) to known polyomaviruses in the late region. The virus was found by PCR in 6 (1%) of 637 nasopharyngeal aspirates and in 1 (0.5%) of 192 fecal samples but was not detected in sets of urine and blood samples. Since polyomaviruses have oncogenic potential and may produce severe disease in immunosuppressed individuals, continued searching for the virus in different medical contexts is important. This finding further illustrates how unbiased screening of respiratory tract samples can be used for the discovery of diverse virus types.

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 (HPV) DNA in tonsillar cancer: clinical correlates, risk of relapse, and survival.

Human papillomavirus (HPV) is more commonly found in tonsillar cancer than in other head and neck cancers. The importance of HPV status in tonsillar cancer for prognosis remains unclear. The aim of the present study was to investigate the frequency of HPV in tonsillar cancer and to correlate the presence of HPV with tumor stage, nodal status, grade of differentiation, risk of relapse, and survival. HPV DNA and HPV type were determined, using PCR, in pre‐treatment biopsies from 60 cases of primary tonsillar cancer. All patients had undergone full‐dose radiotherapy, 45% as the only treatment modality, and 55% in combination with surgery. HPV 16 was detected in 43% (26/60) of the cancers including 1 double infection of both HPV 16 and HPV 33. Patients with HPV+ tonsillar cancer showed less risk of relapse within 3 years after diagnosis, with a better odds ratio of 4.18 as compared with HPV− patients (p = 0.025). Furthermore, cause specific survival was significantly (p = 0.047) better in patients with HPV+ tonsillar carcinomas. At 3 years after diagnosis the survival rate was 65.3% in the HPV+ group and 31.5% in the HPV− group, and at 5 years the survival rate was 53.5% and 31.5%, respectively. The better outcome for patients with HPV+ tonsillar cancer was independent of TNM stage, nodal status, gender and age. These results indicate that HPV status is a significantly favorable prognostic factor in tonsillar cancer and may be used as a marker in order to optimize the treatment of patients with this type of cancer. Int. J. Cancer 89:300–304, 2000.

Human papillomavirus type 16 is episomal and a high viral load may be correlated to better prognosis in tonsillar cancer

The aim of our study was to investigate the physical state and the viral load of HPV‐16 in tonsillar cancer and to correlate these findings with clinical outcome. To distinguish between integrated and episomal forms of HPV, 22 fresh‐frozen tonsillar cancer samples were analysed by a method based on restriction enzyme cleavage, ligation and PCR (rliPCR). HPV‐16 was detected in 11/22 and HPV‐33 in 1/22 of the cancers, hence 12/22 (55%) of the tumours were HPV positive. Only extrachromosomal forms of HPV‐16 were observed. Full‐length episomal HPV was detected exclusively in 7/11 of the cancers, whereas both full‐length and deleted forms of episomal HPV‐16 were found in parallel in 2 other tumours. In 1 tumour only a deleted episomal form of HPV‐16 was present. In the remaining HPV‐16 positive tumour both full‐length episomal as well as an 11 kbp PCR product were detected and if the 11 kbp product contained integrated HPV, or was off‐size linearised episomal could not be determined. In 2 cervical cancer controls, HPV‐16 was integrated and could be chromosome located. HPV‐16 was quantified by real‐time PCR and most tonsillar cancers contained between 10 to a few hundred copies of HPV per β‐actin. The 6 patients with tumour sections with ≥190 HPV‐16 copies/β‐actin remained tumour free (p = 0.026) and had a better survival rate (p = 0.039) when compared to the 5 patients with tumours sections with ≤60 HPV‐16 copies/β‐actin. In conclusion, HPV‐16 is mainly episomal in tonsillar cancer. The viral load showed a wide distribution and the clinical outcome in our study was better when the HPV load was higher.

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.

Oropharyngeal Cancer Epidemic and Human Papillomavirus

Patients with HPV-positive cancer were young and lacked traditional risk factors. Oropharyngeal Cancer Epidemic and HPV

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.

Human papillomavirus is more common in base of tongue than in mobile tongue cancer and is a favorable prognostic factor in base of tongue cancer patients

The frequency of human papilloma virus (HPV) and its influence on clinical outcome was analyzed retrospectively in pre‐treatment paraffin embedded biopsies from 110 patients with tongue cancer. The presence of HPV DNA was examined in 85 mobile tongue tumors and 25 base of tongue tumors by a polymerase chain reaction (PCR) with 2 general primer pairs, GP5+/6+ and CPI/IIG. When HPV‐DNA was found, HPV‐type specific primers and direct sequencing were used for HPV sub‐type verification. Twelve of 110 (10.9%) samples were HPV‐positive; 9 for HPV‐16, 1 for HPV‐33, 1 for HPV‐35 and 1 could not be analyzed because of shortage of DNA. HPV was significantly more common in base of tongue tumors (10/25, 40.0%) compared to tumors of the mobile tongue (2/85, 2.3%). The influence of HPV on clinical outcome in mobile tongue cancer could not be studied, due to that HPV was present in too few cases. Of the 19 patients with base of tongue cancer that were included in the survival analysis, however, 7 patients with HPV‐positive base of tongue cancer had a significantly favorable 5‐year survival rate compared to the 12 HPV‐negative patients. In conclusion, HPV is significantly more common in base of tongue cancer than in mobile tongue cancer, and has a positive impact on disease‐specific survival in patients with base of tongue 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.