Estimating the conditional probability of developing human papilloma virus related oropharyngeal cancer by combining machine learning and inverse Bayesian modelling.

The epidemic increase in the incidence of Human Papilloma Virus (HPV) related Oropharyngeal Squamous Cell Carcinomas (OPSCCs) in several countries worldwide represents a significant public health concern. Although gender neutral HPV vaccination programmes are expected to cause a reduction in the incidence rates of OPSCCs, these effects will not be evident in the foreseeable future. Secondary prevention strategies are currently not feasible due to an incomplete understanding of the natural history of oral HPV infections in OPSCCs. The key parameters that govern natural history models remain largely ill-defined for HPV related OPSCCs and cannot be easily inferred from experimental data. Mathematical models have been used to estimate some of these ill-defined parameters in cervical cancer, another HPV related cancer leading to successful implementation of cancer prevention strategies. We outline a "double-Bayesian" mathematical modelling approach, whereby, a Bayesian machine learning model first estimates the probability of an individual having an oral HPV infection, given OPSCC and other covariate information. The model is then inverted using Bayes' theorem to reverse the probability relationship. We use data from the Surveillance, Epidemiology, and End Results (SEER) cancer registry, SEER Head and Neck with HPV Database and the National Health and Nutrition Examination Surveys (NHANES), representing the adult population in the United States to derive our model. The model contains 8,106 OPSCC patients of which 73.0% had an oral HPV infection. When stratified by age, sex, marital status and race/ethnicity, the model estimated a higher conditional probability for developing OPSCCs given an oral HPV infection in non-Hispanic White males and females compared to other races/ethnicities. The proposed Bayesian model represents a proof-of-concept of a natural history model of HPV driven OPSCCs and outlines a strategy for estimating the conditional probability of an individual's risk of developing OPSCC following an oral HPV infection.

Platelets, immune cells and the coagulation cascade; friend or foe of the circulating tumour cell?

Cancer cells that transit from primary tumours into the circulatory system are known as circulating tumour cells (CTCs). These cancer cells have unique phenotypic and genotypic characteristics which allow them to survive within the circulation, subsequently extravasate and metastasise. CTCs have emerged as a useful diagnostic tool using "liquid biopsies" to report on the metastatic potential of cancers. However, CTCs by their nature interact with components of the blood circulatory system on a constant basis, influencing both their physical and morphological characteristics as well as metastatic capabilities. These properties and the associated molecular profile may provide critical diagnostic and prognostic capabilities in the clinic. Platelets interact with CTCs within minutes of their dissemination and are crucial in the formation of the initial metastatic niche. Platelets and coagulation proteins also alter the fate of a CTC by influencing EMT, promoting pro-survival signalling and aiding in evading immune cell destruction. CTCs have the capacity to directly hijack immune cells and utilise them to aid in CTC metastatic seeding processes. The disruption of CTC clusters may also offer a strategy for the treatment of advance staged cancers. Therapeutic disruption of these heterotypical interactions as well as direct CTC targeting hold great promise, especially with the advent of new immunotherapies and personalised medicines. Understanding the molecular role that platelets, immune cells and the coagulation cascade play in CTC biology will allow us to identify and characterise the most clinically relevant CTCs from patients. This will subsequently advance the clinical utility of CTCs in cancer diagnosis/prognosis.

Prevalence and concordance of oral HPV infections with cervical HPV infections in women referred to colposcopy with abnormal cytology.

BACKGROUND: Considering the shared aetiology of Human Papillomavirus infections in oropharyngeal and cervical cancers and the possible role for sexual transmission, several key aspects of the relationship between cervical and oral infections merit investigation, including prevalence of concomitant oral HPV infection and type-specific concordance with concurrent cervical infections. METHODS: A cross-section study was performed on women referred to colposcopy clinics with cytological abnormalities and a cervical HPV infection. An oral rinse sample was taken from the participants at their baseline visit for HPV testing, and a demographic and risk factor questionnaire was also administered. HPV DNA testing was carried out on the Cobas 4800 platform and extended genotyping was carried out with the INNO-LiPA HPV Genotyping Extra II assay. HPV genotyping was also carried out on the concurrent cervical tissue samples on all women who had a positive oral HPV infection. RESULTS: The prevalence of oral HPV infections was 10.0% (95%CI:5.9-13.7) in the study population. HPV18 was the most frequent genotype (7.0%). Concordant oral and cervical HPV infections were detected in 28.6% of women. Age (p = 0.005) and level of education (p = 0.02) were significantly associated with a prevalent oral HPV infection. CONCLUSION: Concomitant oral HPV infections were present in 10.0% of women referred to colposcopy with a pre-existing cervical HPV infections and cytological abnormalities. Although mild type-specific concordance was observed between oral and cervical HPV infections, findings suggest that infections at these sites may not be independent of each other.

Socio-economic variations in anticipated adverse reactions to testing HPV positive: Implications for the introduction of primary HPV-based cervical screening.

Some cervical cancer screening programmes are replacing cytology with human papillomavirus (HPV) DNA testing as the primary screening test. Concerns have been previously raised around the potential psychosocial impact of testing positive for HPV. We analysed socio-economic variations in anticipated adverse reactions to testing positive for HPV in women of screening age in the general population. A questionnaire was mailed to a random sample of 5553 women aged 20-64 in 2010, selected through primary care in Ireland. This included questions on: socio-economics; HPV knowledge; and women's anticipated adverse psychosocial responses to testing HPV positive (shame, anxiety, stigma and worry). Multivariable linear regression was used to identify socio-economic factors significantly associated with each anticipated adverse reaction. The response rate was 62% (n = 3470). In multivariate analyses, having only attained primary level education were significantly associated with higher mean scores for all four adverse outcomes. Religion was significantly associated with all four adverse outcomes. Age was associated with anxiety and worry; younger women (<30 years) had the highest mean scores. Being married/cohabiting was significantly associated with significantly lower shame and worry scores. Not working was significantly associated with higher mean anxiety and worry scores. Our large population-based survey found significant socio-economic variations in anticipated adverse reactions to testing HPV positive. In order to minimise possible negative impacts on screening uptake and alleviate potential adverse psychological effects of HPV-based screening on women, screening programmes may need to develop specific messages around HPV infection and HPV screening that target certain subgroups of women.

Trends in, and predictors of, anxiety and specific worries following colposcopy: a 12-month longitudinal study.

OBJECTIVE: Little is known about which women are at greatest risk of adverse psychological after-effects following colposcopy. This study examined time trends in, and identified predictors of, anxiety and specific worries over 12 months. METHODS: Women attending two hospital-based colposcopy clinics for abnormal cervical cytology were invited to complete psychosocial questionnaires at 4, 8 and 12 months following colposcopy. General anxiety and screening-specific worries (about cervical cancer, having sex and future fertility) were measured. Generalised estimating equations were used to assess associations between socio-demographic, lifestyle and clinical variables and risk of psychological outcomes. RESULTS: Of 584 women initially recruited, 429, 343 and 303 completed questionnaires at 4, 8 and 12 months, respectively. Screening-specific worries declined significantly over time but were still relatively high at 12 months: 23%, 39% and 18% for worries about cervical cancer, fertility and having sex, respectively. Anxiety remained stable (20%) over time. Risks of cervical cancer worry and anxiety were both almost double in women without private health insurance (cervical cancer worry: OR = 1.80, 95% CI 1.25-2.61; anxiety: OR = 1.84, 95% CI 1.20-2.84). Younger women (<40 years) had higher risk of fertility worries. Non-Irish women had higher risk of anxiety (OR = 2.13, 95% CI 1.13-4.01). CONCLUSIONS: Screening-specific worries declined over time but anxiety remained stable. Notable proportions of women still reported adverse outcomes 12 months following colposcopy, with predictors varying between outcomes. Women in socio-demographically vulnerable groups were at greatest risk of adverse psychological outcomes. This information could inform development of interventions to alleviate psychological distress post-colposcopy.

Identifying novel hypoxia-associated markers of chemoresistance in ovarian cancer.

BACKGROUND: Ovarian cancer is associated with poor long-term survival due to late diagnosis and development of chemoresistance. Tumour hypoxia is associated with many features of tumour aggressiveness including increased cellular proliferation, inhibition of apoptosis, increased invasion and metastasis, and chemoresistance, mostly mediated through hypoxia-inducible factor (HIF)-1α. While HIF-1α has been associated with platinum resistance in a variety of cancers, including ovarian, relatively little is known about the importance of the duration of hypoxia. Similarly, the gene pathways activated in ovarian cancer which cause chemoresistance as a result of hypoxia are poorly understood. This study aimed to firstly investigate the effect of hypoxia duration on resistance to cisplatin in an ovarian cancer chemoresistance cell line model and to identify genes whose expression was associated with hypoxia-induced chemoresistance. METHODS: Cisplatin-sensitive (A2780) and cisplatin-resistant (A2780cis) ovarian cancer cell lines were exposed to various combinations of hypoxia and/or chemotherapeutic drugs as part of a 'hypoxia matrix' designed to cover clinically relevant scenarios in terms of tumour hypoxia. Response to cisplatin was measured by the MTT assay. RNA was extracted from cells treated as part of the hypoxia matrix and interrogated on Affymetrix Human Gene ST 1.0 arrays. Differential gene expression analysis was performed for cells exposed to hypoxia and/or cisplatin. From this, four potential markers of chemoresistance were selected for evaluation in a cohort of ovarian tumour samples by RT-PCR. RESULTS: Hypoxia increased resistance to cisplatin in A2780 and A2780cis cells. A plethora of genes were differentially expressed in cells exposed to hypoxia and cisplatin which could be associated with chemoresistance. In ovarian tumour samples, we found trends for upregulation of ANGPTL4 in partial responders and down-regulation in non-responders compared with responders to chemotherapy; down-regulation of HER3 in partial and non-responders compared to responders; and down-regulation of HIF-1α in non-responders compared with responders. CONCLUSION: This study has further characterized the relationship between hypoxia and chemoresistance in an ovarian cancer model. We have also identified many potential biomarkers of hypoxia and platinum resistance and provided an initial validation of a subset of these markers in ovarian cancer tissues.

The effect of HPV vaccination on the rate of high-grade cytology in 25-year-old women attending cervical screening in Ireland.

INTRODUCTION: Women vaccinated through the initial catch-up HPV vaccination programme (2011/12 to 2013/14) first became eligible for cervical screening in 2019 at age 25. This study aims to examine the changes in detection of HG cytology outcomes in 25-year-olds screened from 2010 to 2022 compared to population data on HPV vaccination in this group. METHODS: This was an ecological-type study. Cytology results from the CervicalCheck database from 2010 to 2022 (High Grade, Low Grade, and No Abnormality Detected) were plotted against data from the National Immunisation Office on the uptake of HPV vaccinations in females from 2010 to 2022. RESULTS: Vaccination rates in the catch-up programme were lower (44-70%) than for routine HPV immunisation at age 12/13 in 2010/11 (81%). The rate of high-grade cytology in 25-year-olds in 2015-2018 was 3.7% of all cytology tests taken in this age group. For the corresponding period from 2019 to 2022 (when vaccinated women were attending screening), the average percentage of HG cytology in 25-year-olds was 1.5%, representing a significant reduction in HG cytology proportions (p < 0.001). CONCLUSION: This study provides early evidence of the potential impact of HPV vaccination on cervical disease in the Republic of Ireland. Despite lower vaccination uptake in the initial catch-up group, we are seeing early signs of the positive protective effect of HPV vaccination in women at the time of their first cervical screening test. Plans to incorporate individual-level HPV vaccination status for women on the cervical screening register will allow more detailed assessment of the impact of HPV vaccination.

Evaluation of the clinical performance of the cobas 4800 HPV test in patients referred for colposcopy.

The clinical performance of the cobas human papillomavirus (HPV) test for detection of high-grade disease in a colposcopy-referred population was compared with that of Hybrid Capture 2 (HC2). The overall agreement between the tests was 92.3%. Clinical sensitivity and specificity for detection of cervical intraepithelial neoplasia grade 2 or greater (CIN2+) were 90.0% and 55.5% for cobas and 90.5% and 50.2% for HC2, respectively. In conclusion, both tests showed comparable performance for detection of CIN2+.

Calibration of the Becton Dickinson FocalPoint GS slide profiler is essential for optimal performance: Avoiding the algorithm supersaturation cascade effect pitfall.

BACKGROUND: The MAVARIC study supported the use of the FocalPoint GS (FPGS) imaging system "No Further Review" (NFR) technology for cervical screening and recommended further investigation. A validation study (Nuttall et al.) was performed by Cervical Screening Wales before implementing the NFR slide reporting technology within the cervical screening program in Wales, United Kingdom. METHOD: A total of 45,317 SurePath liquid-based cytology cervical screening samples were submitted for FPGS scanning within four Welsh cytology laboratories between 2006 and 2011. The study, Computer Assisted Evaluation, Screening and Reporting, involved scanning the slides using the FPGS and comparing the results with manual screening performed under established Cervical Screening Wales protocols. RESULTS: An increased number of abnormal cases presented in the NFR reporting category, significantly greater than that previously encountered. This anomaly resulted in higher false-negative rates with potentially life-changing consequences for the screening participant. Subsequent investigation determined that this increase in cases created an algorithm cascade or "sump" effect, which resulted in an unprecedented increased number of samples categorized as NFR. This exceeded the calibration parameters set for the FPGS and was thought to be caused by an increase in the number of younger women attending for screening following the death of a young reality television celebrity from cervical cancer. CONCLUSION: Adequate and timely calibration of FPGS technology is vital for quality assurance of the results produced, particularly following events that may impact on cervical precancer incidence rates. Failure to do so can result in potentially catastrophic screening incidents that are avoidable.

Plasminogen activator inhibitor 1 is associated with high-grade serous ovarian cancer metastasis and is reduced in patients who have received neoadjuvant chemotherapy.

Introduction: High-grade serous ovarian cancer (HGSOC) is the most prevalent and deadliest subtype of epithelial ovarian cancer (EOC), killing over 140,000 people annually. Morbidity and mortality are compounded by a lack of screening methods, and recurrence is common. Plasminogen-activator-inhibitor 1 (PAI-1, the protein product of SERPIN E1) is involved in hemostasis, extracellular matrix (ECM) remodeling, and tumor cell migration and invasion. Overexpression is associated with poor prognosis in EOC. Platelets significantly increase PAI-1 in cancer cells in vitro, and may contribute to the hematogenous metastasis of circulating tumor cells (CTCs). CTCs are viable tumor cells that intravasate and travel through the circulation-often aided by platelets - with the potential to form secondary metastases. Here, we provide evidence that PAI-1 is central to the platelet-cancer cell interactome, and plays a role in the metastatic cascade. Methods: SK-OV-3 cells where PAI-1 had been silenced, treated with healthy donor platelets, and treated with platelet-conditioned medium were used as an in vitro model of metastatic EOC. Gene expression analysis was performed using RNA-Seq data from untreated cells and cells treated with PAI-1 siRNA or negative control, each with and without platelets. Four cohorts of banked patient plasma samples (n = 239) were assayed for PAI-1 by ELISA. Treatment-naïve (TN) whole blood (WB) samples were evaluated for CTCs in conjunction with PAI-1 evaluation in matched plasma. Results and discussion: Significant phenotypic changes occurring when PAI-1 was silenced and when platelets were added to cells were reflected by RNA-seq data, with PAI-1 observed to be central to molecular mechanisms of EOC metastasis. Increased proliferation was observed in cells treated with platelets. Plasma PAI-1 significantly correlated with advanced disease in a TN cohort, and was significantly reduced in a neoadjuvant chemotherapy (NACT) cohort. PAI-1 demonstrated a trend towards significance in overall survival (OS) in the late-stage TN cohort, and correlation between PAI-1 and neutrophils in this cohort was significant. 72.7% (16/22) of TN patients with plasma PAI-1 levels higher than OS cutoff were CTC-positive. These data support a central role for PAI-1 in EOC metastasis, and highlight PAI-1's potential as a biomarker, prognostic indicator, or gauge of treatment response in HGSOC.

Prediction of Prostate Cancer Biochemical and Clinical Recurrence Is Improved by IHC-Assisted Grading Using Appl1, Sortilin and Syndecan-1.

Gleason scoring is used within a five-tier risk stratification system to guide therapeutic decisions for patients with prostate cancer. This study aimed to compare the predictive performance of routine H&E or biomarker-assisted ISUP (International Society of Urological Pathology) grade grouping for assessing the risk of biochemical recurrence (BCR) and clinical recurrence (CR) in patients with prostate cancer. This retrospective study was an assessment of 114 men with prostate cancer who provided radical prostatectomy samples to the Australian Prostate Cancer Bioresource between 2006 and 2014. The prediction of CR was the primary outcome (median time to CR 79.8 months), and BCR was assessed as a secondary outcome (median time to BCR 41.7 months). The associations of (1) H&E ISUP grade groups and (2) modified ISUP grade groups informed by the Appl1, Sortilin and Syndecan-1 immunohistochemistry (IHC) labelling were modelled with BCR and CR using Cox proportional hazard approaches. IHC-assisted grading was more predictive than H&E for BCR (C-statistic 0.63 vs. 0.59) and CR (C-statistic 0.71 vs. 0.66). On adjusted analysis, IHC-assisted ISUP grading was independently associated with both outcome measures. IHC-assisted ISUP grading using the biomarker panel was an independent predictor of individual BCR and CR. Prospective studies are needed to further validate this biomarker technology and to define BCR and CR associations in real-world cohorts.

Corrigendum to "Trust and cancer screening: Effects of a screening controversy on women's perceptions of cervical cancer screening" [Prev. Med. Rep. 25 (2022) 101684].

[This corrects the article DOI: 10.1016/j.pmedr.2021.101684.].

Trust and cancer screening: Effects of a screening controversy on women's perceptions of cervical cancer screening.

There is a paucity of data on trust of service users in cervical screening. A significant controversy in Ireland's national cervical cancer screening programme emerged in 2018. The Health Service Executive (HSE) confirmed that a clinical audit had revealed that more than 200 women who developed cancer had not been told of earlier misdiagnosed smear tests. During this high profile controversy we conducted qualitative interviews exploring factors that influence cervical screening participation. Women who had been invited for routine screening tests were recruited from the national screening register. Telephone interviews were conducted with 48 women aged 25-65 years; with a range of screening histories - 34 were adequately screened (attended all routine screening tests) and 14 were inadequately screened (attended some/no screening tests). Thematic analysis was conducted and all interviewees spontaneously raised the screening controversy revealing that the crisis had resulted in serious loss of trust, faith and confidence in the screening programme. Publicity surrounding the controversy had some beneficial effects, including increased awareness of the value of screening and beliefs that intense focus on the programme will improve the service long-term. Strategies which incorporate these findings could help rebuild trust in screening.

Development and Validation of a Raman Spectroscopic Classification Model for Cervical Intraepithelial Neoplasia (CIN).

The mortality associated with cervical cancer can be reduced if detected at the precancer stage, but current methods are limited in terms of subjectivity, cost and time. Optical spectroscopic methods such as Raman spectroscopy can provide a rapid, label-free and nondestructive measurement of the biochemical fingerprint of a cell, tissue or biofluid. Previous studies have shown the potential of Raman spectroscopy for cervical cancer diagnosis, but most were pilot studies with small sample sizes. The aim of this study is to show the clinical utility of Raman spectroscopy for identifying cervical precancer in a large sample set with validation in an independent test set. Liquid-based cervical cytology samples (n = 662) (326 negative, 200 cervical intraepithelial neoplasia (CIN)1 and 136 CIN2+) were obtained as a training set. Raman spectra were recorded from single-cell nuclei and subjected to a partial least squares discriminant analysis (PLSDA). In addition, the PLSDA classification model was validated using a blinded independent test set (n = 69). A classification accuracy of 91.3% was achieved with only six of the blinded samples misclassified. This study showed the potential clinical utility of Raman spectroscopy with a good classification of negative, CIN1 and CIN2+ achieved in an independent test set.

The Role of HPV in Determining Treatment, Survival, and Prognosis of Head and Neck Squamous Cell Carcinoma.

Human papillomavirus (HPV) infection has been identified as a significant etiological agent in the development of head and neck squamous cell carcinoma (HNSCC). HPV's involvement has alluded to better survival and prognosis in patients and suggests that different treatment strategies may be appropriate for them. Only some data on the epidemiology of HPV infection in the oropharyngeal, oral cavity, and laryngeal SCC exists in Europe. Thus, this study was carried out to investigate HPV's impact on HNSCC patient outcomes in the Irish population, one of the largest studies of its kind using consistent HPV testing techniques. A total of 861 primary oropharyngeal, oral cavity, and laryngeal SCC (OPSCC, OSCC, LSCC) cases diagnosed between 1994 and 2013, identified through the National Cancer Registry of Ireland (NCRI), were obtained from hospitals across Ireland and tested for HPV DNA using Multiplex PCR Luminex technology based in and sanctioned by the International Agency for Research on Cancer (IARC). Both overall and cancer-specific survival were significantly improved amongst all HPV-positive patients together, though HPV status was only a significant predictor of survival in the oropharynx. Amongst HPV-positive patients in the oropharynx, surgery alone was associated with prolonged survival, alluding to the potential for de-escalation of treatment in HPV-related OPSCC in particular. Cumulatively, these findings highlight the need for continued investigation into treatment pathways for HPV-related OPSCC, the relevance of introducing boys into national HPV vaccination programs, and the relevance of the nona-valent Gardasil-9 vaccine to HNSCC prevention.

Identifying ways to maximise cervical screening uptake: a qualitative study of GPs' and practice nurses' cervical cancer screening-related behaviours.

Background:  Cervical screening uptake is declining in several countries. Primary care practitioners could play a greater role in maximising uptake, but better understanding is needed of practitioners' cervical screening-related behaviours. Among general practitioners (GPs) and practice nurses, we aimed to identify cervical screening-related clinical behaviours; clarify practitioners' roles/responsibilities; and determine factors likely to influence clinical behaviours. Methods: Telephone interviews were conducted with GPs and practice nurses in Ireland. Interview transcripts were analysed using the Theoretical Domains Framework (TDF), a comprehensive psychological framework of factors influencing clinical behaviour. Results: 14 GPs and 19 practice nurses participated. Key clinical behaviours identified were offering smears and encouraging women to attend for smears. Smeartaking responsibility was considered a predominantly female role. Of 12 possible theoretical domains, 11 were identified in relation to these behaviours. Those judged to be the most important were beliefs about capabilities; environmental context and resources; social influences; and behavioural regulation. Difficulties in obtaining smears from certain subgroups of women and inexperience of some GPs in smeartaking arose in relation to beliefs about capabilities. The need for public health education and reluctance of male practitioners to discuss cervical screening with female patients emerged in relation to social influences.   Conclusions: We identified - for the first time - primary care practitioners' cervical-screening related clinical behaviours, their perceived roles and responsibilities, and factors likely to influence behaviours. The results could inform initiatives to enable practitioners to encourage women to have smear tests which in turn, may help increase cervical screening uptake.

Raman Spectroscopy of Liquid-Based Cervical Smear Samples as a Triage to Stratify Women Who Are HPV-Positive on Screening.

The role of persistent high-risk human papillomavirus (HPV) infection in the development of cervical precancer and cancer is now well accepted, and HPV testing has recently been introduced for primary cervical screening. However, the low specificity of HPV DNA testing can result in large numbers of women with an HPV-positive result, and additional triage approaches are needed to avoid over-referral to colposcopy and overtreatment. The aim of this study was to assess Raman spectroscopy as a potential triage test to discriminate between transient and persistent HPV infection. HPV DNA status and mRNA status were confirmed in ThinPrep® cervical samples (n = 60) using the Cobas 4800 and APTIMA HPV test, respectively. Raman spectra were recorded from single-cell nuclei and subjected to partial least squares discriminant analysis (PLSDA). In addition, the PLSDA classification model was validated using a blinded independent test set (n = 14). Sensitivity of 85% and specificity of 92% were achieved for the classification of transient and persistent HPV infection, and this increased to 90% sensitivity and 100% specificity when mean sample spectra were used instead of individual cellular spectra. This study showed that Raman spectroscopy has potential as a triage test for HPV-positive women to identify persistent HPV infection.

Raman spectral cytopathology for cancer diagnostic applications.

Raman spectroscopy can provide a rapid, label-free, nondestructive measurement of the chemical fingerprint of a sample and has shown potential for cancer screening and diagnosis. Here we report a protocol for Raman microspectroscopic analysis of different exfoliative cytology samples (cervical, oral and lung), covering sample preparation, spectral acquisition, preprocessing and data analysis. The protocol takes 2 h 20 min for sample preparation, measurement and data preprocessing and up to 8 h for a complete analysis. A key feature of the protocol is that it uses the same sample preparation procedure as commonly used in diagnostic cytology laboratories (i.e., liquid-based cytology on glass slides), ensuring compatibility with clinical workflows. Our protocol also covers methods to correct for the spectral contribution of glass and sample pretreatment methods to remove contaminants (such as blood and mucus) that can obscure spectral features in the exfoliated cells and lead to variability. The protocol establishes a standardized clinical routine allowing the collection of highly reproducible data for Raman spectral cytopathology for cancer diagnostic applications for cervical and lung cancer and for monitoring suspicious lesions for oral cancer.

Exposure to tobacco smoke measured by urinary nicotine metabolites increases risk of p16/Ki-67 co-expression and high-grade cervical neoplasia in HPV positive women: A two year prospective study.

BACKGROUND: Human papillomavirus (HPV) is considered the strongest epidemiologic risk factor for cervical cancer. However, it is not a sufficient cause given the high prevalence of transient infections. We examined the relationship between exposure to tobacco smoke, measured using urinary nicotine metabolite concentrations, and p16/Ki-67 co-expression in cervical smears and subsequent risk of developing CIN2+/CIN3+ lesions in HPV positive women. METHODS: This prospective longitudinal study enrolled women presenting to colposcopy with cytological abnormalities LSIL/ASCUS at the National Maternity Hospital, Dublin. Women gave a urine sample which was used to perform the Nicotine Metabolite Assay (Siemens). HPV positive (HC2) cervical smears were stained by immunocytochemistry for p16/Ki-67 (CINtec PLUS, Roche). Two year follow-up data, including histological diagnosis, was collected for each woman. Crude and adjusted odds ratios were calculated using logistic regression to investigate associations between tobacco smoke, p16/Ki-67 positivity and CIN2+/CIN3 + . RESULTS: In total, 275 HPV positive women were included. Women with nicotine metabolite concentrations above 500 ng/mL, indicative of smoking, were classified as smokers. Smokers were at an increased risk of testing positive for p16/Ki-67 (OR 1.678; 1.027-2.740) and CIN2+ and CIN3+ (OR 1.816; 1.107-2.977 and OR 2.453; 1.200-5.013) in compared to non-smokers. In p16/Ki-67 positive women, smoking further increased their risk of CIN2+/CIN3+ (OR 2.290; 1.017-5.159 and OR 3.506 (1.534-8.017). CONCLUSION: HPV positive women exposed to tobacco smoke are at a higher risk of testing positive for p16/Ki-67 co-expression. Risk of high-grade disease is almost doubled in women who are exposed to tobacco smoke.

Gene expression profiling in cervical cancer: identification of novel markers for disease diagnosis and therapy.

Cervical cancer, a potentially preventable disease, remains the second most common malignancy in women worldwide. Human papillomavirus is the single most important etiological agent in cervical cancer. HPV contributes to neoplastic progression through the action of two viral oncoproteins E6 and E7, which interfere with critical cell cycle pathways, p53, and retinoblastoma. However, evidence suggests that HPV infection alone is insufficient to induce malignant changes and other host genetic variations are important in the development of cervical cancer. Advances in molecular biology and high throughput gene expression profiling technologies have heralded a new era in biomarker discovery and identification of molecular targets related to carcinogenesis. These advancements have improved our understanding of carcinogenesis and will facilitate screening, early detection, management, and personalised targeted therapy. In this chapter, we have described the use of high density microarrays to assess gene expression profiles in cervical cancer. Using this approach we have identified a number of novel genes which are differentially expressed in cervical cancer, including several genes involved in cell cycle regulation. These include p16ink4a, MCM 3 and 5, CDC6, Geminin, Cyclins A-D, TOPO2A, CDCA1, and BIRC5. We have validated expression of mRNA using real-time PCR and protein by immunohistochemistry.