PET-CT Surveillance versus Neck Dissection in Advanced Head and Neck Cancer.

BACKGROUND: The role of image-guided surveillance as compared with planned neck dissection in the treatment of patients with squamous-cell carcinoma of the head and neck who have advanced nodal disease (stage N2 or N3) and who have received chemoradiotherapy for primary treatment is a matter of debate. METHODS: In this prospective, randomized, controlled trial, we assessed the noninferiority of positron-emission tomography-computed tomography (PET-CT)-guided surveillance (performed 12 weeks after the end of chemoradiotherapy, with neck dissection performed only if PET-CT showed an incomplete or equivocal response) to planned neck dissection in patients with stage N2 or N3 disease. The primary end point was overall survival. RESULTS: From 2007 through 2012, we recruited 564 patients (282 patients in the planned-surgery group and 282 patients in the surveillance group) from 37 centers in the United Kingdom. Among these patients, 17% had nodal stage N2a disease and 61% had stage N2b disease. A total of 84% of the patients had oropharyngeal cancer, and 75% had tumor specimens that stained positive for the p16 protein, an indicator that human papillomavirus had a role in the causation of the cancer. The median follow-up was 36 months. PET-CT-guided surveillance resulted in fewer neck dissections than did planned dissection surgery (54 vs. 221); rates of surgical complications were similar in the two groups (42% and 38%, respectively). The 2-year overall survival rate was 84.9% (95% confidence interval [CI], 80.7 to 89.1) in the surveillance group and 81.5% (95% CI, 76.9 to 86.3) in the planned-surgery group. The hazard ratio for death slightly favored PET-CT-guided surveillance and indicated noninferiority (upper boundary of the 95% CI for the hazard ratio, <1.50; P=0.004). There was no significant difference between the groups with respect to p16 expression. Quality of life was similar in the two groups. PET-CT-guided surveillance, as compared with neck dissection, resulted in savings of £1,492 (approximately $2,190 in U.S. dollars) per person over the duration of the trial. CONCLUSIONS: Survival was similar among patients who underwent PET-CT-guided surveillance and those who underwent planned neck dissection, but surveillance resulted in considerably fewer operations and it was more cost-effective. (Funded by the National Institute for Health Research Health Technology Assessment Programme and Cancer Research UK; PET-NECK Current Controlled Trials number, ISRCTN13735240.).

Human papillomavirus prevalence and type-distribution in cervical glandular neoplasias: Results from a European multinational epidemiological study.

Cervical glandular neoplasias (CGN) present a challenge for cervical cancer prevention due to their complex histopathology and difficulties in detecting preinvasive stages with current screening practices. Reports of human papillomavirus (HPV) prevalence and type-distribution in CGN vary, providing uncertain evidence to support prophylactic vaccination and HPV screening. This study [108288/108290] assessed HPV prevalence and type-distribution in women diagnosed with cervical adenocarcinoma in situ (AIS, N = 49), adenosquamous carcinoma (ASC, N = 104), and various adenocarcinoma subtypes (ADC, N = 461) from 17 European countries, using centralised pathology review and sensitive HPV testing. The highest HPV-positivity rates were observed in AIS (93.9%), ASC (85.6%), and usual-type ADC (90.4%), with much lower rates in rarer ADC subtypes (clear-cell: 27.6%; serous: 30.4%; endometrioid: 12.9%; gastric-type: 0%). The most common HPV types were restricted to HPV16/18/45, accounting for 98.3% of all HPV-positive ADC. There were variations in HPV prevalence and ADC type-distribution by country. Age at diagnosis differed by ADC subtype, with usual-type diagnosed in younger women (median: 43 years) compared to rarer subtypes (medians between 57 and 66 years). Moreover, HPV-positive ADC cases were younger than HPV-negative ADC. The six years difference in median age for women with AIS compared to those with usual-type ADC suggests that cytological screening for AIS may be suboptimal. Since the great majority of CGN are HPV16/18/45-positive, the incorporation of prophylactic vaccination and HPV testing in cervical cancer screening are important prevention strategies. Our results suggest that special attention should be given to certain rarer ADC subtypes as most appear to be unrelated to HPV.

PATHOS: a phase II/III trial of risk-stratified, reduced intensity adjuvant treatment in patients undergoing transoral surgery for Human papillomavirus (HPV) positive oropharyngeal cancer.

BACKGROUND: Human papillomavirus-positive oropharyngeal squamous cell carcinoma is increasing in incidence worldwide. Current treatments are associated with high survival rates but often result in significant long-term toxicities. In particular, long-term dysphagia has a negative impact on patient quality of life and health. The aim of PATHOS is to determine whether reducing the intensity of adjuvant treatment after minimally invasive transoral surgery in this favourable prognosis disease will result in better long-term swallowing function whilst maintaining excellent disease-specific survival outcomes. METHODS/DESIGN: The study is a multicentre phase II/III randomised controlled trial for patients with biopsy-proven Human papillomavirus-positive oropharyngeal squamous cell cancer staged T1-T3 N0-N2b with a primary tumour that is resectable via a transoral approach. Following transoral surgery and neck dissection, patients are allocated into three groups based on pathological risk factors for recurrence. Patients in the low-risk pathology group will receive no adjuvant treatment, as in standard practice. Patients in the intermediate-risk pathology group will be randomised to receive either standard dose post-operative radiotherapy (control) or reduced dose radiotherapy. Patients in the high-risk pathology group will be randomised to receive either post-operative chemoradiotherapy (control) or radiotherapy alone. The primary outcome of the phase II study is patient reported swallowing function measured using the MD Anderson Dysphagia Inventory score at 12 months post-treatment. If the phase II study is successful, PATHOS will proceed to a phase III non-inferiority trial with overall survival as the primary endpoint. DISCUSSION: PATHOS is a prospective, randomised trial for Human papillomavirus-positive oropharyngeal cancer, which represents a different disease entity compared with other head and neck cancers. The trial aims to demonstrate that long-term dysphagia can be lessened by reducing the intensity of adjuvant treatment without having a negative impact on clinical outcome. The study will standardise transoral surgery and post-operative intensity-modulated radiotherapy protocols in the UK and develop a gold-standard swallowing assessment panel. An associated planned translational research programme, underpinned by tumour specimens and sequential blood collected as part of PATHOS, will facilitate further empirical understanding of this new disease and its response to treatment. TRIAL REGISTRATION: This study is registered with ClinicalTrials.gov identifier NCT02215265 .

Activity, safety, and feasibility of cidofovir and imiquimod for treatment of vulval intraepithelial neoplasia (RT³VIN): a multicentre, open-label, randomised, phase 2 trial.

BACKGROUND: Vulval intraepithelial neoplasia is a skin disorder affecting the vulva that, if left untreated, can become cancerous. Currently, the standard treatment for patients with vulval intraepithelial neoplasia is surgery, but this approach does not guarantee cure and can be disfiguring, causing physical and psychological problems, particularly in women of reproductive age. We aimed to assess the activity, safety, and feasibility of two topical treatments--cidofovir and imiquimod--as an alternative to surgery in female patients with vulval intraepithelial neoplasia. METHODS: We recruited female patients (age 16 years or older) from 32 centres to an open-label, randomised, phase 2 trial. Eligibility criteria were biopsy-proven vulval intraepithelial neoplasia grade 3 and at least one lesion that could be measured accurately. We randomly allocated patients to topical treatment with either 1% cidofovir (supplied as a gel in a 10 g tube, to last 6 weeks) or 5% imiquimod (one 250 mg sachet for every application), to be self-applied three times a week for a maximum of 24 weeks. Randomisation (1:1) was done by stratified minimisation via a central computerised system, with stratification by hospital, disease focality, and presentation stage. The primary endpoint was a histologically confirmed complete response at the post-treatment assessment visit 6 weeks after the end of treatment (a maximum of 30 weeks after treatment started). Analysis of the primary endpoint was by intention to treat. Secondary outcomes were toxic effects (to assess safety) and adherence to treatment (to assess feasibility). We present results after all patients had reached the primary endpoint assessment point at 6 weeks; 2-year follow-up of complete responders continues. This trial is registered with Current Controlled Trials, ISRCTN 34420460. FINDINGS: Between Oct 21, 2009, and Jan 11, 2013, 180 participants were enrolled to the study; 89 patients were randomly allocated cidofovir and 91 were assigned imiquimod. At the post-treatment assessment visit, a complete response had been achieved by 41 (46%; 90% CI 37·0-55·3) patients allocated cidofovir and by 42 (46%; 37·2-55·3) patients assigned imiquimod. After 6 weeks of treatment, 156 (87%) patients (78 in each group) had adhered to the treatment regimen. Five patients in the cidofovir group and seven in the imiquimod group either withdrew or were lost to follow-up before the first 6-week safety assessment. Adverse events of grade 3 or higher were reported in 31 (37%) of 84 patients allocated cidofovir and 39 (46%) of 84 patients assigned imiquimod; the most frequent grade 3 and 4 events were pain in the vulva, pruritus, fatigue, and headache. INTERPRETATION: Cidofovir and imiquimod were active, safe, and feasible for treatment of vulval intraepithelial neoplasia and warrant further investigation in a phase 3 setting. Both drugs are effective alternatives to surgery for female patients with vulval intraepithelial neoplasia after exclusion of occult invasive disease. FUNDING: Cancer Research UK.

Human papillomavirus infection is rare in nonmalignant tonsil tissue in the UK: implications for tonsil cancer precursor lesions.

The incidence of human papillomavirus (HPV)-associated tonsil cancer is increasing but the prevalence of HPV, and of premalignant precursors, in tonsil tissue is unknown. We aimed to assess prevalence of HPV infection in nonmalignant tonsillar crypt epithelia and to histopathologically characterise positive samples. Formalin-fixed paraffin-embedded (FFPE) tonsil tissue specimens were obtained from an age- and sex-stratified random sample of patients aged 0-69 years whose paired tonsils were archived following elective tonsillectomy at hospitals throughout England and Southern Scotland from 2004 to 2008. Homogenised fresh-frozen tonsil tissue was also obtained from archive for two random subsets of males aged 25-34 and over 44. HPV status was assessed in all samples for 20 mucosal HPV types by GP5+/6+ polymerase chain reaction (PCR) enzyme immunoassay and by HPV16 type-specific PCR targeting the E6 gene. In the homogenised material, HPV status was also assessed for 44 HPV types by SPF10-PCR enzyme immunoassay. Of 4,095 randomly sampled FFPE specimens, amplifiable DNA was extracted from 3,377 (82.5%) and from 511 of 524 (97.5%) homogenised tonsils. HPV DNA was identified in 0 of 3,377 (0%, 95% CI 0-0.089%) fixed samples and 0 of 511 (0%, 95% CI 0-0.58%) homogenised samples. This suggests HPV infection may be rare in tonsil reticulated crypt epithelia. Furthermore, we found no evidence of HPV-associated premalignant neoplasia. These data suggest that if HPV-associated premalignant lesions do occur, they are likely to be rare and may have a high risk of progression to carcinoma.

mRNA sequencing of novel cell lines from human papillomavirus type-16 related vulval intraepithelial neoplasia: consequences of expression of HPV16 E4 and E5.

Vulval intraepithelial neoplasia is a precursor of vulval cancer and is commonly caused by infection with Human Papillomavirus (HPV). Development of topical treatments for vulval intraepithelial neoplasia requires appropriate in vitro models. This study evaluated the feasibility of primary culture of vulval intraepithelial neoplasia biopsy tissue to produce cell lines for use as in vitro models. A potentially immortal cell line was produced which gave rise to three monoclonal lines. These lines were characterized for HPV genomic integration and for viral gene expression using ligation-mediated PCR and quantitative PCR. Distinct patterns of viral integration and gene expression were observed among the three lines. Integration and expression data were validated using deep sequencing of mRNA. Gene ontology analyses of these data also demonstrated that expression of the HPV16 E4 and E5 proteins resulted in substantial changes in the composition of the cell membrane and extracellular space, associated with alterations in cell adhesion and differentiation. These data illustrate the diverse patterns of HPV gene expression potentially present within a single lesion. The derived cell lines provide useful models to investigate the biology of vulval intraepithelial neoplasia and the interactions between different HPV gene products and potential therapeutic agents.

HPV16 L1 and L2 DNA methylation predicts high-grade cervical intraepithelial neoplasia in women with mildly abnormal cervical cytology.

DNA methylation changes in human papillomavirus type 16 (HPV16) DNA are common and might be important for identifying women at increased risk of cervical cancer. Using recently published data from Costa Rica we developed a classification score to differentiate women with cervical intraepithelial neoplasia grade 2 or 3 (CIN2/3) from those with no evident high-grade lesions. Here, we aim to investigate the performance of the score using data from the UK. Exfoliated cervical cells at baseline and 6-months follow-up were analyzed in 84 women selected from a randomized clinical trial of women undergoing surveillance for low-grade cytology. Selection of women for the methylation study was based on detectable HPV16 in the baseline sample. Purified DNA was bisulfite converted, amplified and pyrosequenced at selected CpG sites in the viral genome (URR, E6, L1 and L2), with blinding of laboratory personnel to the clinical data. The primary measure was a predefined score combining the mean methylation in L1 and any methylation in L2. At the second follow-up visit, 73/84 (87%) women were HPV16 positive and of these 25 had a histopathological diagnosis of CIN2/3. The score was significantly associated with CIN2/3 (area under curve = 0.74, p = 0.002). For a cutoff with 92% sensitivity, colposcopy could have been avoided in 40% (95% CI 27-54%) of HPV16 positive women without CIN2/3; positive predictive value was 44% (32-58%) and negative predictive value was 90% (71-97%). We conclude that quantitative DNA methylation assays could help to improve triage among HPV16 positive women.

Differences in human papillomavirus type distribution in high-grade cervical intraepithelial neoplasia and invasive cervical cancer in Europe.

Knowledge of differences in human papillomavirus (HPV)-type prevalence between high-grade cervical intraepithelial neoplasia (HG-CIN) and invasive cervical cancer (ICC) is crucial for understanding the natural history of HPV-infected cervical lesions and the potential impact of HPV vaccination on cervical cancer prevention. More than 6,000 women diagnosed with HG-CIN or ICC from 17 European countries were enrolled in two parallel cross-sectional studies (108288/108290). Centralised histopathology review and standardised HPV-DNA typing were applied to formalin-fixed paraffin-embedded cervical specimens dated 2001-2008. The pooled prevalence of individual HPV types was estimated using meta-analytic methods. A total of 3,103 women were diagnosed with HG-CIN and a total of 3,162 with ICC (median ages: 34 and 49 years, respectively), of which 98.5 and 91.8% were HPV-positive, respectively. The most common HPV types in women with HG-CIN were HPV16/33/31 (59.9/10.5/9.0%) and in ICC were HPV16/18/45 (63.3/15.2/5.3%). In squamous cell carcinomas, HPV16/18/33 were most frequent (66.2/10.8/5.3%), and in adenocarcinomas, HPV16/18/45 (54.2/40.4/8.3%). The prevalence of HPV16/18/45 was 1.1/3.5/2.5 times higher in ICC than in HG-CIN. The difference in age at diagnosis between CIN3 and squamous cervical cancer for HPV18 (9 years) was significantly less compared to HPV31/33/'other' (23/20/17 years), and for HPV45 (1 year) than HPV16/31/33/'other' (15/23/20/17 years). In Europe, HPV16 predominates in both HG-CIN and ICC, whereas HPV18/45 are associated with a low median age of ICC. HPV18/45 are more frequent in ICC than HG-CIN and associated with a high median age of HG-CIN, with a narrow age interval between HG-CIN and ICC detection. These findings support the need for primary prevention of HPV16/18/45-related cervical lesions.

Decreased HPV-specific T cell responses and accumulation of immunosuppressive influences in oropharyngeal cancer patients following radical therapy.

Oropharyngeal cancer (OPC) is a type of squamous cell head and neck cancer that is often associated with human papillomavirus (HPV) infection, suggesting the potential for immunotherapeutic targeting of HPV antigens. This study aimed to determine the effect of radical therapy on HPV-specific T cells and other immune parameters in 20 OPC patients, as a prelude to future immunotherapy studies. HPV DNA could be detected in 9/12 available tissue samples (8/9 HPV(+) samples were also p16(+)). HPV-specific T cell responses against HPV16 E6 and E7 peptides were detected by enzyme-linked immunoSPOT in 10/13 and 8/13 evaluable patients, respectively, but did not appear to correlate with HPV status. Post-treatment, both HPV E6 and E7 T cell responses were decreased (4/13 and 2/13 patients, respectively). These reductions in T cell response could not be explained by a concurrent decrease in memory T cells whose absolute numbers were relatively unaffected by radical therapy (27,975 vs. 25,661/10(5) PBMC) despite a significant decrease in overall lymphocyte counts (1.74 vs. 0.69 × 10(9)/L). Instead, there were significant increases in regulatory T cells (3.7 vs. 6.8 %) and a population of myeloid-derived suppressor cells (CD14(-)HLA-DR(-)CD15(hi), 12.38 vs. 21.92 %). This suggests that immunosuppression may contribute to the reduction in HPV-specific T cell responses post-treatment, although study of larger patient cohorts will be required to test whether this affects clinical outcome. Overall these findings suggest that HPV-targeted immunotherapy in post-therapy OPC patients will require multiple strategies to boost T cell immunity and to overcome the influence of immunosuppressive cells.

Human Papillomavirus-associated oropharyngeal cancer: an observational study of diagnosis, prevalence and prognosis in a UK population.

BACKGROUND: The incidence of Human Papillomavirus (HPV) associated oropharyngeal cancer (OPC) is increasing. HPV-associated OPC appear to have better prognosis than HPV-negative OPC. The aim of this study was to robustly determine the prevalence of HPV-positive OPC in an unselected UK population and correlate HPV positivity with clinical outcome. METHODS: HPV testing by GP5+/6+ PCR, In Situ Hybridisation (ISH) and p16 immunohistochemistry (IHC) was performed on 138 OPCs diagnosed in South Wales (UK) between 2001-06. Kaplan-Meier analysis was used to correlate HPV status with clinical outcome. RESULTS: Using a composite definition of HPV positivity (HPV DNA and p16 overexpression), HPV was detected in 46/83 (55%) samples where DNA quality was assured. Five year overall survival was 75.4% (95% CI: 65.2 to 85.5) in HPV-positives vs 25.3% (95% CI: 14.2 to 36.4) in HPV negatives, corresponding to a 78% reduction in death rate (HR 0.22, p < 0.001). HPV-positives had less locoregional recurrence but second HPV-positive Head and Neck primaries occurred. Poor quality DNA in fixed pathological specimens reduced both HPV prevalence estimates and the prognostic utility of DNA-based HPV testing methods. As a single marker, p16 was least affected by sample quality and correlated well with prognosis, although was not sufficient on its own for accurate HPV prevalence reporting. CONCLUSIONS: This study highlights the significant burden of OPC associated with HPV infection. HPV positive cases are clinically distinct from other OPC, and are associated with significantly better clinical outcomes. A composite definition of HPV positivity should be used for accurate prevalence reporting and up-front DNA quality assessment is recommended for any DNA-based HPV detection strategy.

Combined HPV 16 E2 and L1 methylation predict response to treatment with cidofovir and imiquimod in patients with vulval intraepithelial neoplasia.

BACKGROUND: Topical cidofovir and imiquimod can effectively treat approximately 55% of patients with vulval intraepithelial neoplasia (VIN), thus avoiding the need for surgery. Human papillomavirus (HPV) E⁢2 gene methylation predicts response to treatment but a methylation measurement is only obtainable in approximately 50% of patients. OBJECTIVE: This work aimed to determine if the applicability and predictive power of the E⁢2 methylation assay could be improved by combining it with the components of a host and viral DNA methylation panel (S5) that has been found to predict disease progression in patients with cervical intraepithelial neoplasia. METHODS: HPV E2 methylation and S5 classifier score were measured in fresh tissue samples collected pre-treatment from 132 patients with biopsy-proven VIN grade 3 who participated in a multicentre clinical trial and were randomised to treatment with cidofovir or imiquimod. RESULTS: Combining HPV16 E⁢2 and HPV16 L⁢1 methylation provides a biomarker that is both predictive of response to topical treatment and that can produce a clinically applicable result for all patients. Patients with HPV 16 L⁢1^high and HPV 16 E⁢2^high (36/132 (27.3%)) were more likely to respond to treatment with cidofovir (12/15 (80.0%)) than imiquimod (9/21 (42.9%)) (p= 0.026). Patients with HPV 16 L⁢1^low or HPV 16 E⁢2^low (including those with no HPV/unassessable methylation) were more likely to respond to imiquimod: 23/50 (46.0%) vs 31/46 (67.4%) (p= 0.035). CONCLUSIONS: Combined HPV E⁢2 and L⁢1 methylation is a potential predictive marker in treatment for all patients with VIN. These findings justify validation in a prospective trial.

The risk of cervical cancer associated with specific types of human papillomavirus: a case-control study in a UK population.

Mounting evidence supports incorporation of HPV testing into cervical screening; however, the optimal test format and target population have yet to be confirmed. Assessment of the potential benefits of type-specific testing requires estimation of the risk associated with infection with individual HPV types. However, the risk posed by individual HPV types may be population specific and influenced by cervical screening practice. The existing data on HPV type-specific risk is derived largely from unscreened populations. Our study addressed the lack of data on HPV type-specific risk in cytologically screened populations using a case-control study of 262 invasive cervical cancers diagnosed in Wales between 2000 and 2006, and 8,428 controls who attended for cytological screening in 2004. The analysis showed that the odds ratios (ORs) for infection with HPV 16 and 18 are considerable; 2770 (95% CI 1050-7320) and 950 (95% CI 330-2740), respectively, and that the OR for other oncogenic types are in general considerably less (ranging, where quantified, from 20.2 to 386 in the same population). The effect of age on OR associated with particular HPV types was also assessed; this indicated that infection with a high-risk HPV in women older than 40 years was associated with an approximately 30-fold increased risk of invasive cervical cancer relative to women younger than 40 years. These results indicate that there is significant prognostic information associated with knowledge of HPV type.

Human papillomavirus type distribution in vulval intraepithelial neoplasia determined using PapilloCheck DNA Microarray.

Vulval intraepithelial neoplasia is a precursor of vulval carcinoma, and is frequently associated with human papillomavirus (HPV) infection. Estimates of HPV prevalence in vulval intraepithelial neoplasia vary widely in the UK. The objective of this study was to assess HPV infection in a sample of women with vulval intraepithelial neoplasia, confirmed histologically, and determine the proportion of disease associated with HPV types targeted by prophylactic HPV vaccines. HPV infection was assessed in biopsies from 59 patients using the Greiner Bio-One PapilloCheck® DNA chip assay. Valid results were obtained for 54 cases. HPV infection was present in 43 of the 54 cases (79.6%: 95% CI 67.1-88.2%). The most common HPV types were HPV 16 (33/54: 61.1%), HPV 33 (8/54: 14.8%), HPV 6 (5/54: 9.3%), and HPV 42 (3/54: 5.6%). The mean age of HPV positive women was significantly less than the mean age of HPV negative women. This is the largest UK series of vulval intraepithelial neoplasia in which HPV type has been investigated, and 34/54 (63.0%, 95% CI: 49.6-78.6%) cases were associated with HPV 16/18, which are targeted by current prophylactic HPV vaccines.

Sensitivity of human papillomavirus­positive and ­negative oropharyngeal cancer cell lines to ionizing irradiation.

Human papillomavirus­positive (HPV+) oropharyngeal squamous cell carcinoma (OPSCC) has increased in incidence and has a much better prognosis than HPV­negative (HPV­) OPSCC with radiotherapy alone, but exactly why is unknown. The present study therefore aimed to further examine the sensitivity and possible changes in gene expression of several HPV+ and HPV­ OPSCC, including various novel cell lines, upon ionizing irradiation (IR). Previously established HPV+ UM­SCC­47, UPCI­SCC­90, CU­OP­2, CU­OP­3 and HPV­ UM­SCC­4, UM­SCC­6, UM­SCC­74a, UM­SCC­19 and newly established CU­OP­17 and CU­OP­20, characterised here, were subjected to 0­6 Gy. Surviving fractions of each cell line were tested by clonogenic assays, and irregularities in cell cycle responses were examined by flow cytometry, while changes in gene expression were followed by mRNA sequencing. HPV+ OPSCC cell lines showed greater variation in sensitivity to ionizing irradiation (IR) and tended to be more sensitive than HPV­ OPSCC cell lines. However, their IR sensitivity was not correlated to the proportion of cells in G2 arrest, and HPV­ cell lines generally showed lower increases in G2 after IR. Upon IR with 2 Gy, mRNA sequencing revealed an increase in minor HPV integration sites in HPV+ cell lines, and some changes in gene expression in OPSCC cell lines, but not primarily those associated with DNA repair. To conclude, HPV+ OPSCC cell lines showed greater variation in their sensitivity to IR, with some that were radioresistant, but overall the HPV+ OPSCC group still tended to be more sensitive to IR than the HPV­ OPSCC group. In addition, HPV+ OPSCC lines were more frequently in G2 as compared to HPV­ cell lines, but the increase in G2 arrest upon IR in HPV+ OPSCC was not correlated to sensitivity to IR. Increases in minor HPV integration sites and changes in gene expression were also demonstrated after irradiation with 2 Gy.

The potential impact of human papillomavirus vaccination in contemporary cytologically screened populations may be underestimated: an observational retrospective analysis of invasive cervical cancers.

The aim of this study was to determine the proportion of invasive cervical cancers attributable to human papillomavirus (HPV) types 16 and 18 in a contemporary, cytologically well-screened UK population. This was achieved in a retrospective observational analysis by HPV typing 453 archival invasive cervical cancers diagnosed between January 1, 2000 and September 1, 2006. Pathological material was collected from 9 hospitals across Wales (UK), and HPV typing and pathology review was conducted at a central laboratory. Genotyping for high-risk HPV DNA was performed by PCR-enzyme immunoassay using the GP5+/6+ primer set. DNA was successfully extracted from 297 cases. Two hundred and eighty cases were included in the final analysis. The proportion of cases which had only HPV 16 and/or 18 was 219 of 280 (78.2%, 95% CI = 73.0-82.7); the proportion of cases which had HPV 16 or 18 and another HPV type was 230 of 280 (82.1%, 95% CI = 77.2-86.2). The proportion of cervical cancers associated with infection with HPV types 16 and 18 has previously been estimated at around 70%. The appropriate figure for a cytologically well-screened UK population appears to be approximately 80%. Hence, the potential impact of the current vaccination programme may be underestimated.

Sensitivity to inhibition of DNA repair by Olaparib in novel oropharyngeal cancer cell lines infected with Human Papillomavirus.

The incidence of Human Papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCC) is increasing rapidly in the UK. Patients with HPV-positive OPSCC generally show superior clinical responses relative to HPV-negative patients. We hypothesised that these superior responses could be associated with defective repair of DNA double strand breaks (DSB). The study aimed to determine whether defective DNA repair could be associated with sensitivity to inhibition of DNA repair using the PARP inhibitor Olaparib. Sensitivity to Olaparib, and induction and repair of DNA damage, were assessed in a panel of 8 OPSCC cell-lines, including 2 novel HPV-positive lines. Effects on cell cycle distribution and levels of PARP1 and p53 were quantified. RNA-sequencing was used to assess differences in activity of DNA repair pathways. Two HPV-positive OPSCC lines were sensitive to Olaparib at potentially therapeutic doses (0.1-0.5 µM). Two HPV-negative lines were sensitive at an intermediate dose. Four other lines, derived from HPV-positive and HPV-negative tumours, were resistant to PARP inhibition. Only one cell-line, UPCISCC90, showed results consistent with the original hypothesis i.e. that in HPV-positive cells, treatment with Olaparib would cause accumulation of DSB, resulting in cell cycle arrest. There was no evidence that HPV-positive tumours exhibit defective repair of DSB. However, the data suggest that a subset of OPSCC may be susceptible to PARP-inhibitor based therapy.

Human Papillomavirus DNA Methylation Predicts Response to Treatment Using Cidofovir and Imiquimod in Vulval Intraepithelial Neoplasia 3.

Purpose: Response rates to treatment of vulval intraepithelial neoplasia (VIN) with imiquimod and cidofovir are approximately 57% and 61%, respectively. Treatment is associated with significant side effects and, if ineffective, risk of malignant progression. Treatment response is not predicted by clinical factors. Identification of a biomarker that could predict response is an attractive prospect. This work investigated HPV DNA methylation as a potential predictive biomarker in this setting.Experimental Design: DNA from 167 cases of VIN 3 from the RT3 VIN clinical trial was assessed. HPV-positive cases were identified using Greiner PapilloCheck and HPV 16 type-specific PCR. HPV DNA methylation status was assessed in three viral regions: E2, L1/L2, and the promoter, using pyrosequencing.Results: Methylation of the HPV E2 region was associated with response to treatment. For cidofovir (n = 30), median E2 methylation was significantly higher in patients who responded (P ≤ 0.0001); E2 methylation >4% predicted response with 88.2% sensitivity and 84.6% specificity. For imiquimod (n = 33), median E2 methylation was lower in patients who responded to treatment (P = 0.03; not significant after Bonferroni correction); E2 methylation <4% predicted response with 70.6% sensitivity and 62.5% specificity.Conclusions: These data indicate that cidofovir and imiquimod may be effective in two biologically defined groups. HPV E2 DNA methylation demonstrated potential as a predictive biomarker for the treatment of VIN with cidofovir and may warrant investigation in a biomarker-guided clinical trial. Clin Cancer Res; 23(18); 5460-8. ©2017 AACR.

Recovery of human papillomavirus nucleic acids from liquid-based cytology media.

Residual material from liquid-based cytology (LBC) samples, collected during cervical screening, is a valuable resource for molecular biological analysis. Because of the central role played by human papillomavirus (HPV) in the development of cervical cancer, assays are being developed to quantify HPV gene expression in LBC material. Using quantitative realtime PCR we have compared recovery of HPV DNA and RNA from two of the most widely used LBC systems (the ThinPrep system (Cytyc Corp.) and the SurePath system (TriPath Imaging, Inc.)). Recovery of RNA was unaffected by storage in ThinPrep media, however storage of cells in SurePath resulted in significantly reduced yields (between 10(4)- and 10(8)-fold reduction depending on extraction technique). Given the increasing prominence and importance of molecular diagnostics and prognostics this is an important finding, and must be considered in relation to choice of LBC system.

Geographic variation in human papillomavirus-related oropharyngeal cancer: Data from 4 multinational randomized trials.

BACKGROUND: There are variations in the proportions of head and neck cancers caused by the human papillomavirus (HPV) between countries and regions. It is unclear if these are true variations or due to different study designs and assays. METHODS: We tested formalin-fixed paraffin-embedded diagnostic biopsies for p16 immunohistochemistry and HPV-DNA (by polymerase chain reaction [PCR] and in situ hybridization [ISH]) using validated protocols on samples from 801 patients with head and neck cancer recruited prospectively between 2006 and 2011 in 4 randomized controlled trials (RCTs). RESULTS: Twenty-one percent of patients (170 of 801) showed both HPV-DNA and p16-positivity, detected almost exclusively in oropharyngeal cancer (55%; 15 of 302); and only 1% of the patients (5 of 499) with nonoropharyngeal cancer were HPV positive. HPV-positive oropharyngeal cancer differed between Western and Eastern Europe (37%, 155 of 422 vs 6%, 8 of 144; p < .0001) and between Western Europe and Asia (37% vs 2%; 4 of 217; p < .0001). Other independent determinants of HPV positivity were tumor site and smoking. CONCLUSION: This is the first study to establish geographic variability as an independent risk factor in HPV-positive oropharyngeal cancer prevalence, with higher prevalence in Western Europe. © 2016 The Authors Head & Neck Published by Wiley Periodicals, Inc. Head Neck 38: E1863-E1869, 2016.

HPV-Related Oropharynx Cancer in the United Kingdom: An Evolution in the Understanding of Disease Etiology.

A rising incidence of oropharyngeal squamous cell carcinoma (OPSCC) incidence has occurred throughout the developed world, where it has been attributed to an increasing impact of human papillomavirus (HPV) on disease etiology. This report presents the findings of a multicenter cross-sectional retrospective study aimed at determining the proportion of HPV-positive and HPV-negative OPSCC within the United Kingdom. Archival tumor tissue blocks from 1,602 patients previously diagnosed with OPSCC (2002-2011) were collated from 11 centers. HPV status was determined with three validated commercial tests to provide valid data for 1,474 cases in total. Corresponding national incidence data from the same decade were obtained from UK Cancer registries. The overall proportion of HPV+ OPSCC between 2002 and 2011 was 51.8% [95% confidence interval (CI), 49.3-54.4], and this remained unchanged throughout the decade [unadjusted RR = 1.00 (95% CI, 0.99-1.02)]. However, over the same period, the incidence of OPSCC in the broader UK population underwent a 2-fold increase [age-standardized rate 2002: 2.1 (95% CI, 1.9-2.2); 2011: 4.1 (95% CI, 4.0-4.3)]. Although the number of OPSCCs diagnosed within the United Kingdom from 2002 to 2011 nearly doubled, the proportion of HPV+ cases remained static at approximately 50%. Our results argue that the rapidly increasing incidence of OPSCC in the United Kingdom cannot be solely attributable to the influence of HPV. The parallel increase in HPV+ and HPV- cases we documented warrants further investigation, so that appropriate future prevention strategies for both types of disease can be implemented. Cancer Res; 76(22); 6598-606. ©2016 AACR.