Torbjörn Ramqvist

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.

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

Taxonomical developments in the family Polyomaviridae

The Polyomaviridae Study Group of the International Committee on Taxonomy of Viruses (ICTV) has recommended several taxonomical revisions, as follows: The family Polyomaviridae, which is currently constituted as a single genus (Polyomavirus), will be comprised of three genera: two containing mammalian viruses and one containing avian viruses. The two mammalian genera will be designated Orthopolyomavirus and Wukipolyomavirus, and the avian genus will be named Avipolyomavirus. These genera will be created by the redistribution of species from the current single genus (Polyomavirus) and by the inclusion of several new species. In addition, the names of several species will be changed to reflect current usage.

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.

Vaccination, immune and gene therapy based on virus-like particles against viral infections and cancer

Virus-like particles (VLPs) are self-assembling, non-replicating particles lacking the viral genome that are formed by one or several viral structural proteins. VLPs can be purified after expression in yeast cells, insect cells using baculoviruses, Escherichia coli or mammalian cells. Recently, vaccines based on VLPs have come into focus with the FDA approval of a VLP-based vaccine against human papilloma viruses. However, this application of VLPs is just one of many developments within the VLP field. Other potential applications under development besides vaccines against viruses or cancers also include gene delivery and treatment of different disorders.

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.