Studies on human papillomavirus and molecular markers in head-neck cancer

Background. Oropharyngeal squamous cell carcinoma (OSCC), where tonsillar and base of tongue cancer (TSCC and BOTSCC) dominate, is associated with smoking and alcohol as well as human papillomavirus (HPV) infection. The incidence of TSCC and BOTSCC, has increased lately, mainly due to HPV infection. In addition, patients with HPV-positive TSCC and BOTSCC have a better clinical outcome compared to those with the corresponding HPV-negative tumors (80% vs. 40% 5 year disease free survival (DFS)). Recently, head and neck cancer treatment has been intensified with chemotherapy and more intensive radiotherapy. This is likely unnecessary for 80% of HPV-positive TSCC and BOTSCC patients. Aims. Due to the increase in TSCC and BOTSCC, we wanted to follow oral HPV-prevalence in healthy sexually active youth as well as in patients with TSCC and BOTSCC. This in order to disclose e.g. whether there were specific traits of oral HPV infection in the latter group. In addition, the presence of different HPV16 E6 variants in TSCC were analyzed as well as new biomarkers, which could aid in the identification of patients with HPV positive TSCC and BOTSCC that could be eligible for de-escalated treatment. Result. In Paper I we showed that oral HPV prevalence was 9.3% among youth attending a youth clinic in Stockholm. Oral HPV infection was more common in women with genital infection and there was also HPV-type concordance between the oral and cervical sites. When testing samples from patients with suspected HNSCC in Paper II, nearly all HPVDNA+ oral samples were derived from patients with HPVDNA+ TSCC and BOTSCC. For healthy subjects with oral HPVDNA+ infection, the relative viral load was very low. In Paper III we found that the HPV16E6 L83V variant was common in TSCC, cervical cancer (CC) and cervical samples (CS), while the rare HPV16E6 R10G variant was present in a proportion of TSCC, but absent in CC and only sporadic in CS samples. Neither L83V nor R10G had any significant impact on clinical outcome. In paper IV, high number of CD8+ tumor infiltrating lymphocytes (TILs) was correlated to a better clinical outcome, especially for patients with HPVDNA+ and HPVDNA+ /p16 positive TSCC and BOTSCC. CD4+ TIL counts were not linked to clinical outcome or survival for patients with HPVDNA+ tumors, although there was a tendency of better survival for patients with HPVDNA- and HPVDNA-/p16-negative tumors. Finally in Paper V, patients with HPVDNA+ TSCC and BOTSCC and absent/weak as compared to medium/ strong CD44 intensity staining had a significantly better 3-year DFS and overall survival. Conclusion. Oral HPV infection was relatively frequent in Stockholm youth as compared to other studies during the same time period, but the relative viral load was in general lower than that found for patients with HPV-positive TSCC and BOTSCC. HPV16E6 L83V variant was common in TSCC, CC and CS, while the R10G variant was present in a proportion of TSCC, but absent in CC and only sporadic in CS samples. Both high CD8+ TIL infiltration and absent/weak CD44 intensity staining seemed to be promising predictive markers for patients with HPVDNA+ TSCC and BOTSCC

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 (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 an

Reduced Expression of the Antigen Processing Machinery Components TAP2, LMP2, and LMP7 in Tonsillar and Base of Tongue Cancer and Implications for Clinical Outcome

OBJECTIVES: Patients with human papillomavirus (HPV)–positive tonsillar squamous cell carcinoma (TSCC) and base of tongue squamous cell carcinoma (BOTSCC) have a better clinical outcome than those with corresponding HPV-negative tumors. Moreover, there is a strong positive correlation between absent/low as opposed to strong HLA class I expression and favorable clinical outcome for HPV-positive tumors, while the reverse applies to HPV-negative tumors. The expression of the antigen processing machinery (APM) components TAP1, TAP2, LMP2, and LMP7 in these tumors in relation to HPV status, HLA class I expression, each other, and clinical outcome was therefore investigated. MATERIAL AND METHODS: Formalin-fixed paraffin-embedded TSCC and BOTSCC, derived from 151 patients and previously analyzed for HPV DNA, HLA class I, and LMP10 expression were stained by immunohistochemistry for TAP1, TAP2, LMP2, and LMP7. RESULTS: Absent/low TAP2, LMP2, and LMP7 expression, similar to HLA class I and LMP10, was common in TSCC and BOTSCC, irrespective of HPV status. Expression of TAP1 and TAP2 was correlated, as was LMP2 to LMP7. LMP2 and LMP7 expression was also associated to HLA class I expression. Moreover, absence of LMP7 was linked to increased disease-free survival in both HPV-positive and HPV-negative cases. CONCLUSION: Reduced expression of TAP2, LMP2, and LMP7 was frequent in TSCC and BOTSCC and their expression as well as that of TAP1 was often interrelated. Furthermore, low LMP7 expression correlated to better clinical outcome and may, together with HPV status, potentially be used for prediction of treatment response

Correlation of LMP10 expression and clinical outcome in Human Papillomavirus (HPV) positive and HPV-Negative tonsillar and base of tongue cancer.

To examine LMP10 expression and its possible impact on clinical outcome in human papillomavirus (HPV) positive and HPV-negative tonsillar and base of tongue squamous cell carcinoma (TSCC and BOTSCC).Outcome is better in HPV-positive TSCC and BOTSCC compared to matching HPV-negative tumours, with roughly 80% vs. 40% 5-year disease free survival (DFS) with less aggressive treatment than today's chemoradiotherapy. Since current treatment often results in harmful side effects, less intensive therapy, with sustained patient survival would be an attractive alternative. However, other markers together with HPV status are necessary to select patients and for this purpose LMP10 expression is investigated here in parallel to HPV status and clinical outcome.From 385 patients diagnosed between 2000 and 2007 at the Karolinska University Hospital, 278 formalin fixed paraffin embedded TSCC and BOTSCC biopsies, with known HPV DNA status, were tested for LMP10 nuclear and cytoplasmic expression (fraction of positive cells and staining intensity). The data was then correlated to clinical outcome.An absent/low compared to a moderate/high LMP10 nuclear fraction of positive cells was correlated to a better 3-year DFS in the HPV-positive group of patients (log-rank p = 0.005), but not in the HPV-negative group. In the HPV-negative group of patients, in contrast to the HPV-positive group, moderate/high LMP10 cytoplasmic fraction and weak/moderate/high LMP10 cytoplasmic intensity correlated to a better 3-year DFS (p = 0.003 and p = 0.001) and 3-year overall survival (p = 0.001 and 0.009).LMP10 nuclear expression in the HPV-positive group and LMP10 cytoplasmic expression in the HPV-negative group of patients correlated to better clinical outcome

Absent/weak CD44 intensity and positive human papillomavirus (HPV) status in oropharyngeal squamous cell carcinoma indicates a very high survival

Patients with human papillomavirus DNA positive (HPVDNA+) oropharyngeal squamous cell carcinoma (OSCC) have better clinical outcome than those with HPV DNA negative (HPVDNA-) OSCC upon intensive oncological treatment. All HPVDNA+ OSCC patients may not require intensive treatment, however, but before potentially deintensifying treatment, additional predictive markers are needed. Here, we examined HPV, p16(INK4a), and CD44 in OSCC in correlation to clinical outcome. Pretreatment tumors from 290 OSCC patients, the majority not receiving chemotherapy, were analyzed for HPV DNA by Luminex and for p16(INK4a) and CD44 by immunohistochemistry. 225/290 (78%) tumors were HPVDNA+ and 211/290 (73%) overexpressed p16(INK4a), which correlated to presence of HPV (P < 0.0001). Presence of HPV DNA, absent/weak CD44 intensity staining correlated to favorable 3-year disease-free survival (DFS) and overall survival (OS) by univariate and multivariate analysis, and likewise for p16(INK4a) by univariate analysis. Upon stratification for HPV, HPVDNA+ OSCC with absent/weak CD44 intensity presented the significantly best 3-year DFS and OS, with >95% 3-year DFS and OS. Furthermore, in HPVDNA+ OSCC, p16(INK4a)+ overexpression correlated to a favorable 3-year OS. In conclusion, patients with HPVDNA+ and absent/weak CD44 intensity OSCC presented the best survival and this marker combination could possibly be used for selecting patients for tailored deintensified treatment in prospective clinical trials. Absence of/weak CD44 or presence of human papillomavirus (HPV) DNA was shown as a favorable prognostic factors in tonsillar and tongue base cancer. Moreover, patients with the combination of absence of/weak CD44 and presence of HPV DNA presented a very favorable outcome. Therefore, we suggest that this marker combination could potentially be used to single out patients with a high survival that could benefit from a de-escalated oncological treatment

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

Flow chart showing the tumors from patients diagnosed with TSCC in the County of Stockholm 2000–2007 and the TSCC selected for inclusion in the study.

<p>Flow chart showing the tumors from patients diagnosed with TSCC in the County of Stockholm 2000–2007 and the TSCC selected for inclusion in the study.</p

BRIP1, PALB2, and RAD51C mutation analysis reveals their relative importance as genetic susceptibility factors for breast cancer

Mutations in the recognized breast cancer susceptibility genes BRCA1, BRCA2, TP53, ATM, and CHEK2 account for approximately 20% of hereditary breast cancer. This raises the possibility that mutations in other biologically relevant genes may be involved in genetic predisposition to breast cancer. In this study, BRIP1, PALB2, and RAD51C were sequenced for mutations as a result of previously being associated with breast cancer risk due to their role in the double-strand break repair pathway and their close association with BRCA1 and BRCA2. Two truncating mutations in PALB2 (Q66X and W1038X), one of which is has not been reported before, were detected in an independent Australian cohort of 70 individuals with breast or ovarian cancer, and have strong family histories of breast or breast/ovarian cancer. In addition, six missense variants predicted to be causative were detected, one in BRIP1 and five in PALB2. No causative variants were identified in RAD51C. This study supports recent observations that although rare, PALB2 mutations are present in a small but substantial proportion of inherited breast cancer cases, and indicates that RAD51C at a population level does not account for a substantial number of familial breast cancer cases