Lars Sivars

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.

Human papillomavirus and p53 expression in cancer of unknown primary in the head and neck region in relation to clinical outcome.

Patients with cancer of unknown primary (CUP) in the head neck region are generally treated with neck dissection followed by radiotherapy at times combined with chemotherapy, a treatment associated with considerable side effects. Some of these tumors may originate as human papillomavirus (HPV)‐positive oropharyngeal squamous cell carcinoma (OSCC), with better clinical outcome than head neck squamous cell cancer (HNSCC) in general, and could potentially do well with less treatment. Here, we therefore investigated whether HPV status and p53‐expression correlated to clinical outcome in patients with CUP in the head neck region. Fifty metastases were analyzed for presence of HPV DNA, and expression of p16INK4A and p53 and the data were correlated to clinical outcome. Patients with HPV DNA‐positive (HPVDNA+) metastases had significantly better 5‐year overall survival (OS) compared to those with HPVDNA− metastases (80.0% vs. 36.7%, respectively; P = 0.004), with a similar tendency for disease‐free survival (DFS). These survival rates showed excellent concordance with those of HPVDNA+ and HPVDNA− OSCC in Sweden during the same time period, strengthening the hypothesis that HPVDNA+ head and neck CUP may originate from HPVDNA+ OSCC. In addition, having absent/intermediary‐low as compared to high expression of p53 correlated to a better prognosis with a 69% as compared to 14% 5‐year OS, respectively (P < 0.001), and for DFS the tendency was analogous. In conclusion, both HPV status and p53 expression are valuable prognostic factors in patients with CUP in the head and neck region and should be further explored for clinical use.

Oral human papillomavirus prevalence in high school students of one municipality in Sweden

Abstract The rise in human papillomavirus (HPV) infection has been suggested to be responsible for the increased incidence of oropharyngeal cancer in the Western world. This has boosted interest in oral HPV prevalence and whether HPV vaccines can prevent oral HPV infection. In a previous study we showed oral HPV prevalence to be almost 10% in youth aged 15–23 y attending a youth clinic in Stockholm, Sweden. However, this may not be a generalizable sample within the Swedish population. Therefore, mouthwashes were used to investigate oral HPV prevalence in 335 Swedish high school students aged 17–21 y (median age 18 y), from 1 municipality with 140,000 inhabitants. The presence of HPV DNA in the oral samples, as examined by a Luminex-based assay, was significantly lower in this cohort, only 1.8% (3.1% in females and 0.6% in males), as compared to our previous study.

A model for predicting clinical outcome in patients with human papillomavirus-positive tonsillar and base of tongue cancer.

AIM To combine clinical and molecular markers into an algorithm for predicting outcome for individual patients with human papillomavirus (HPV) DNA/p16(INK4a) positive tonsillar and base of tongue squamous cell carcinoma (TSCC and BOTSCC). BACKGROUND Head-neck cancer treatment has become more intensified, comprising not only surgery and radiotherapy, but also induction/concomitant chemotherapy and targeted therapy. With less treatment, 3-year disease free survival (DFS) is 80% for HPV-positive TSCC and BOTSCC. An 85-100% 3-year DFS is observed for HPV(+) TSCC and BOTSCC with absence of HLA class I, or CD44 expression, or high CD8(+) tumour-infiltrating lymphocyte (TIL) counts suggesting that therapy could be tapered for many if patients could be identified individually. PATIENTS AND METHODS Patients treated curatively, with HPV DNA/p16(INK4a) positive tumours examined for HLA class I and II, CD44 and CD8(+)TILs, were included. An L1-regularised logistic regression was used to evaluate the effect of the biomarker data, age, stage, diagnosis, smoking and treatment on 3-year risk of death or relapse on a training cohort of 197 patients diagnosed 2000-2007 and validated on a cohort of 118 patients diagnosed 2008-2011. RESULTS The variables finally included in the model were HLA class I, CD8(+) TILs, age, stage and diagnosis (TSCC or BOTSCC). The model showed acceptable discrimination and calibration. The discriminative ability of the model did not diminish after validation (AUC=0.77). CONCLUSION To our knowledge, this is the first model to utilise information from several markers to predict an individual probability of clinical outcome for patients with HPV DNA/p16(INK4a) positive tumours.

Human papillomavirus is a favourable prognostic factor in cancer of unknown primary in the head and neck region and in hypopharyngeal cancer (Review)

Human papillomavirus (HPV), in addition to smoking and alcohol, is a cause of oropharyngeal squamous cell carcinoma (OPSCC), particularly of the tonsils and base of the tongue (TSCC and BOTSCC, respectively). Moreover, HPV-positive TSCC and BOTSCC are associated with a better outcome compared with their HPV-negative counterparts (80 vs. 40% 3-year disease-free survival rate, respectively) and their incidence has increased in several countries. Recently, accumulating evidence of HPV in a considerable proportion of cancers of unknown primary (CUP) in the head and neck region and in a small proportion of hypopharyngeal SCCs has been reported. Furthermore, HPV-positive tumours, particularly cases with HPV DNA positivity in combination with overexpression of p16, also tend to have a better clinical outcome compared with that of the corresponding HPV-negative tumours. This finding is particularly prominent in HPV-positive CUPs of the head and neck region, where the primary tumour likely originates from the oropharynx. Thus, the determination of HPV status and p16 expression may be of value for the diagnosis and treatment of CUP of the head and neck region and may also be of value for hypopharyngeal cancers in the future. However, for hypopharyngeal cancer as well as other non-OPSCCs, additional studies per subsite on the effect of HPV status on survival are required.

A model using concomitant markers for predicting outcome in human papillomavirus positive oropharyngeal cancer

OBJECTIVE Head-neck cancer therapy has become intensified. With radiotherapy alone, 3-year disease-free survival (DFS) is 80% for HPV-positive TSCC/BOTSCC and better for patients with favorable characteristics, suggesting therapy could be tapered for some, decreasing side-effects. Therefore, we built a model to predict progression-free survival for patients with HPV-positive TSCC and BOTSCC. MATERIAL AND METHODS TSCC/BOTSCC patients treated curatively between 2000 and 2011, with HPV16 DNA/E7 mRNA positive tumors examined for CD8+ TILs, HPV16 mRNA and HLA class I expression were included. Patients were split randomly 65/35 into training and validation sets, and LASSO regression was used to select a model in the training set, the performance of which was evaluated in the validation set. RESULTS 258 patients with HPV DNA/E7 mRNA positive tumors could be included, 168 and 90 patients in the respective sets. No treatment improved prognosis compared to radiotherapy alone. CD8+ TIL counts and young age were the strongest predictors of survival, followed by T-stage <3 and presence of HPV16 E2 mRNA. The model had an area under curve (AUC) of 76%. A model where the presence of three of four of these markers defined good prognosis captured 56% of non-relapsing patients with a positive predictive value of 98% in the validation set. Furthermore, the model identified 35% of our cohort that was overtreated and could safely have received de-escalated therapy. CONCLUSION CD8+ TIL counts, age, T-stage and E2 expression could predict progression-free survival, identifying patients eligible for randomized trials with milder treatment, potentially reducing side effects without worsening prognosis.

Human papillomavirus DNA detection in fine-needle aspirates as indicator of human papillomavirus-positive oropharyngeal squamous cell carcinoma: A prospective study.

Human papillomavirus (HPV)‐positive oropharyngeal squamous cell carcinoma (SCC) has a better outcome than most head neck squamous cell carcinomas (HNSCCs) and an HPV‐positive lymph node metastasis likely has an HPV‐positive oropharyngeal SCC origin. Determining HPV‐status in cervical lymph nodes by fine‐needle aspiration cytology (FNAC) may be useful for diagnosis.

Targeted sequencing of tonsillar and base of tongue cancer and human papillomavirus positive unknown primary of the head and neck reveals prognostic effects of mutated FGFR3

BACKGROUND Human papillomavirus positive (HPV+) tonsillar cancer (TSCC), base of tongue cancer (BOTSCC) and unknown primary cancer of the head and neck (HNCUP) have better outcome than corresponding HPV− cancers. To find predictive markers for response to treatment, and correlations and differences in mutated oncogenes and suppressor genes between HPV+ TSCC/BOTSSCC and HPV+ HNCUP and HPV− TSCC/BOTSCC targeted next-generation sequencing was performed of frequently mutated regions in 50 cancer related genes. PATIENTS AND METHODS DNA from 348 TSCC/BOTSCC and 20 HNCUP from patients diagnosed 2000-2011, was sequenced by the Ion Proton sequencing platform using the Ion AmpliSeq Cancer Hotspot Panel v2 to identify frequently mutated regions in 50 cancer related genes. Ion Torrent Variant Caller software was used to call variants. RESULTS 279 HPV+ TSCC/BOTSCC, 46 HPV− TSCC/BOTSCC and 19 HPV+ HNCUP samples qualified for further analysis. Mutations/tumor were fewer in HPV+ TSCC/BOTSCC and HNCUP, compared to HPV− tumors (0.92 vs. 1.32 vs. 1.68). Differences in mutation frequency of TP53 and PIK3CA were found between HPV+ TSCC/BOTSCC and HNCUP and HPV− TSCC/BOTSCC. In HPV+ TSCC/BOTSCC presence of FGFR3 mutations correlated to worse prognosis. Other correlations to survival within the groups were not disclosed. CONCLUSIONS In HPV+ TSCC/BOTSCC mutation of PIK3CA was most frequently observed, while TP53 mutations dominated in HPV− TSCC/BOTSCC. In HPV+ TSCC/BOTSCC and HNCUP, mutations/tumor were similar in frequency and fewer compared to that in HPV− TSCC/BOTSCC. Notably, FGFR3 mutations in HPV+ TSCC/BOTSCC indicated worse prognosis.