The natural history of cervical HPV infection: unresolved issues.

The identification of high-risk human papillomavirus (HPV) types as a necessary cause of cervical cancer offers the prospect of effective primary prevention and the possibility of improving the efficiency of cervical screening programmes. However, for these opportunities to be realized, a more complete understanding of the natural history of HPV infection, and its relationship to the development of epithelial abnormalities of the cervix, is required. We discuss areas of uncertainty, and their possible effect on disease prevention strategies.

A comparison of patient and tumour characteristics in two UK bladder cancer cohorts separated by 20 years.

OBJECTIVES: To compare patient and tumour characteristics at presentation from two large bladder cancer cohorts, with recruitment separated by 15-20 years To identify significant differences in the West Midlands' urothelial cancer of the bladder (UCB) population during this period. PATIENTS AND METHODS: Data were collected prospectively from 1478 patients newly diagnosed with UCB in the West Midlands from January 1991 to June 1992 (Cohort 1), and from 1168 patients newly diagnosed with UBC within the same region from December 2005 to April 2011 (Cohort 2). Gender, age, smoking history, and tumour grade, stage, type, multiplicity and size at presentation were compared using a Pearson chi-square test or Cochran-Armitage trend test, as appropriate. RESULT: Cohort 2 had a higher proportion of male patients (P = 0.021), elderly patients (P < 0.001), grade 3 tumours (P < 0.001), Ta/T1 tumours (P = 0.008), multiple tumours (P < 0.001), and tumours of ≤2 cm in diameter (P < 0.001). CONCLUSIONS: There were significant differences between the cohorts. These differences are potentially explained by an ageing population, changes in grading practices, improved awareness of important symptoms, improved cystoscopic technology, and reductions in treatment delays. Regional cohorts remain important for identifying changes in tumour and patient characteristics that may influence disease management in the UK and beyond.

Sample size calculations for designing clinical proteomic profiling studies using mass spectrometry.

In cancer clinical proteomics, MALDI and SELDI profiling are used to search for biomarkers of potentially curable early-stage disease. A given number of samples must be analysed in order to detect clinically relevant differences between cancers and controls, with adequate statistical power. From clinical proteomic profiling studies, expression data for each peak (protein or peptide) from two or more clinically defined groups of subjects are typically available. Typically, both exposure and confounder information on each subject are also available, and usually the samples are not from randomized subjects. Moreover, the data is usually available in replicate. At the design stage, however, covariates are not typically available and are often ignored in sample size calculations. This leads to the use of insufficient numbers of samples and reduced power when there are imbalances in the numbers of subjects between different phenotypic groups. A method is proposed for accommodating information on covariates, data imbalances and design-characteristics, such as the technical replication and the observational nature of these studies, in sample size calculations. It assumes knowledge of a joint distribution for the protein expression values and the covariates. When discretized covariates are considered, the effect of the covariates enters the calculations as a function of the proportions of subjects with specific attributes. This makes it relatively straightforward (even when pilot data on subject covariates is unavailable) to specify and to adjust for the effect of the expected heterogeneities. The new method suggests certain experimental designs which lead to the use of a smaller number of samples when planning a study. Analysis of data from the proteomic profiling of colorectal cancer reveals that fewer samples are needed when a study is balanced than when it is unbalanced, and when the IMAC30 chip-type is used. The method is implemented in the clippda package and is available in R at: http://www.bioconductor.org/help/bioc-views/release/bioc/html/clippda.html.

Oncogenic human papillomavirus imposes an instructive pattern of DNA methylation changes which parallel the natural history of cervical HPV infection in young women.

The contribution of early virus-induced epigenetic changes to human papillomavirus (HPV)-associated carcinogenesis is poorly understood. Using genome-wide methylation array profiling and a cell-based model, which supports replication of HPV episomes, we found that transfection of primary human foreskin keratinocytes with episomal forms of high-risk HPV types was followed by upregulation of the DNA methyltransferases, DNMT1 and DNMT3B, and changes in the methylation status of cellular genes many of which are reported to be differentially methylated in cervical neoplasia. HPV16- and HPV18-associated changes were not randomly distributed across the genome, but clustered at specific chromosomal locations which mapped on to known HPV integration sites and to chromosomal regions lost and gained in high-grade cervical neoplasia. Methylation changes were directed in part by the same cis-acting factors that appear to direct methylation changes in cancer, the presence of a bivalent chromatin mark in human embryonic stem cells and promoter CpG content; these associations explain much of the ontological profile of genes found to have increased methylation following HPV16 transfection. We were also able to show, using sequential samples from a cohort of young women with incident HPV16 infections, that the detection in cervical samples of methylated forms of the tumour suppressor gene, RARB, often parallels the natural history of cervical HPV infection. Our findings suggest that further investigation of the distribution and determinants of early virus-induced epigenetic reprogramming will provide important insights into the pathogenesis of virus-associated malignancy.

A phase I/II clinical trial in localized prostate cancer of an adenovirus expressing nitroreductase with CB1954 [correction of CB1984].

We report a phase I/II clinical trial in prostate cancer (PCa) using direct intraprostatic injection of a replication defective adenovirus vector (CTL102) encoding bacterial nitroreductase (NTR) in conjunction with systemic prodrug CB1954. One group of patients with localized PCa scheduled for radical prostatectomy received virus alone, prior to surgery, in a dose escalation to establish safety, tolerability, and NTR expression. A second group with local failure following primary treatment received virus plus prodrug to establish safety and tolerability. Based on acceptable safety data and indications of prostate-specific antigen (PSA) responses, an extended cohort received virus at a single dose level plus prodrug. The vector was well tolerated with minimal side effects, had a short half-life in the circulation, and stimulated a robust antibody response. Immunohistochemistry of resected prostate demonstrated NTR staining in tumor and glandular epithelium at all dose levels [5 x 10(10)-1 x 10(12) virus particles (vp)]. A total of 19 patients received virus plus prodrug and 14 of these had a repeat treatment; minimal toxicity was observed and there was preliminary evidence of change in PSA kinetics, with an increase in the time to 10% PSA progression in 6 out of 18 patients at 6 months.

Role of sexual behavior in the acquisition of asymptomatic Epstein-Barr virus infection: a longitudinal study.

BACKGROUND: The natural history of Epstein-Barr virus (EBV) infection is poorly defined. We report the prevalence and subsequent incidence of EBV infection in a cohort of sexually active young women and explore the social and sexual determinants of incident infections. METHODS: The study population was drawn from a cohort of young women, who were recruited for a longitudinal study of risk factors for early cervical neoplasia. A case-control analysis, nested within the cohort of 45 women for whom the first EBV sample tested was EBV-negative and who had further follow-up, was undertaken. EBV serostatus was determined in serum with a synthetic peptide-based enzyme-linked immunosorbent assay; EBV DNA was measured in cervical smears with the use of quantitative polymerase chain reaction. RESULTS: Of 1023 women 15-19 years of age included in this analysis, 978 (95.6%) tested positive for antibodies to EBV in their first serum sample. Of 45 women who tested negative, 22 subsequently acquired an asymptomatic EBV infection; the median time to seroconversion was 25 months (range, 1-60 months), and the median age at seroconversion was 18 years (range, 16-21 years). The risk of seroconversion increased with increasing number of sexual partners [compared with 1 partner, odds ratio (OR) was 1.28 for 2 partners and 2.23 for 3 or more; chiTREND 5.02; df 1; P < 0.05] and was greatest when a new sexual partner had been acquired in the 2 years before seroconversion (OR 4.78; chi 4.62; df 1; P < 0.05). EBV DNA was detected in 9 of 14 women who seroconverted and who also provided cervical samples. CONCLUSIONS: In susceptible young women, the acquisition of EBV infection is associated with their sexual behavior.

A Comparative Analysis of the Influence of Gender, Pathway Delays, and Risk Factor Exposures on the Long-term Outcomes of Bladder Cancer.

BACKGROUND: The relationship between pathway delays and bladder cancer-specific survival is complex because of the influence of tumour- and patient-specific factors. OBJECTIVE: To investigate the influence of tumour factors, patient factors, carcinogen exposure, and pathway delays on the long-term outcome of urothelial bladder cancer (UBC). DESIGN, SETTING, AND PARTICIPANTS: A cohort of 1537 UBC patients were enrolled between January 1, 1991 and June 30, 1992 and followed up for 17.7 yr. The period from the onset of symptoms to first treatment (transurethral resection of bladder tumour, TURBT) was divided into three components of potential delay. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Associations between patient factors, tumour factors, and delay times were analysed using the Pearson χ2 test and the Mann-Whitney U test. Survival was calculated from date of TURBT to date of death or censor date (December 31, 2010). Competing risks of death were assessed with the cumulative incidence function (CIF); CIF comparisons were performed using the Gray test. RESULTS AND LIMITATIONS: At censor, reliable data were available for 1478 patients, of whom 75% had died. Females presented more commonly with muscle-invasive bladder cancer (MIBC; 30% vs 26%) and less frequently with pT1 disease (18% vs 24%; p=0.06) and had a longer total delay time (median 120 d vs 106 d, p=0.02), and those with MIBC had a significantly higher cumulative incidence of death due to UBC (80% vs 67% at 17 yr; p<0.02). Cox regression identified age, smoking status, and tumour stage, grade, and size as the most significant determinants of poor outcome. We did not capture downstream delays associated with cystectomy or radiotherapy. CONCLUSIONS: Female UBC patients present later than males, and our data suggest that delay in referral may be contributory. The relationship between gender, outcomes, delays, and UBC aetiology is complex. PATIENT SUMMARY: We followed a large group of bladder cancer patients for more than 17 yr. The relationship between pathway delays and survival is complex. However, female patients present later than male patients, and our data suggest that delay in referral from general practice may be contributory.

Implementation of RCGP guidelines for acute low back pain: a cluster randomised controlled trial.

BACKGROUND: The Royal College of General Practitioners (RCGP) has produced guidelines for the management of acute low back pain in primary care. AIM: To investigate the impact on patient management of an educational strategy to promote these guidelines among general practitioners (GPs). DESIGN OF STUDY: Group randomised controlled trial, using the health centre as the unit of randomisation. SETTING: Primary care teams in north-west England. METHOD: Twenty-four health centres were randomly allocated to an intervention or control arm. Practices in the intervention arm were offered outreach visits to promote national guidelines on acute low back pain, as well as access to fast-track physiotherapy and to a triage service for patients with persistent symptoms. RESULTS: Twenty-four centres were randomised. Two thousand, one hundred and eighty-seven eligible patients presented with acute low back pain during the study period: 1049 in the intervention group and 1138 in the control group. There were no significant differences between study groups in the proportion of patients who were referred for X-ray, issued with a sickness certificate, prescribed opioids or muscle relaxants, or who were referred to secondary care, but significantly more patients in the intervention group were referred to physiotherapy or the back pain unit (difference in proportion = 12.2%, 95% confidence interval [CI] = 2.8% to 21.6%). CONCLUSION: The management of patients presenting with low back pain to primary care was mostly unchanged by an outreach educational strategy to promote greater adherence to RCGP guidelines among GPs. An increase in referral to physiotherapy or educational programmes followed the provision of a triage service.

Assessment of high-throughput high-resolution MALDI-TOF-MS of urinary peptides for the detection of muscle-invasive bladder cancer.

PURPOSE: There is a need for better biomarkers to both detect bladder cancer and distinguish muscle-invasive (stage T2+) from non-invasive (stage Ta/T1) disease. We assess whether MALDI-TOF-MS of the urine peptidome can achieve this. EXPERIMENTAL DESIGN: We analysed urine from 751 patients with bladder cancer and 127 patients without bladder cancer. Endogenous peptide profiling was performed using a Bruker Ultraflextreme MALDI-TOF-MS. RESULTS: Significant differences were seen between the spectra of urine from patients with and without T2+ disease. Albumin, total protein and haematuria were also elevated in T2+ patients. Haematuria was detected in 39% of patients with Ta/T1 disease and in 77% of patients with T2+ disease. Class prediction models based on MALDI data produced areas under receiver-operator characteristic curves of up to 0.76 but did not significantly outperform a model based on total protein alone. Many peptides significantly associated with invasive disease are fragments of abundant blood proteins and are also associated with haematuria. CONCLUSIONS AND CLINICAL RELEVANCE: Microscopic haematuria is strongly associated with invasive disease; even traces of blood/plasma strongly influence the urinary peptidome. This needs to be taken into consideration when using 'omic' methods to search for urinary biomarkers as blood proteins may give false-positive results.

Narrow-band imaging flexible cystoscopy: a new user's experience.

BACKGROUND AND PURPOSE: We and others have demonstrated previously an improved detection rate for bladder cancer recurrences with narrow-band imaging (NBI) flexible cystoscopy when compared with conventional white-light imaging (WLI) flexible cystoscopy. We investigated whether a "new user" of NBI flexible cystoscopy, previously unfamiliar with this technique, could reproduce these results. PATIENTS AND METHODS: The same protocol from our previous study was used by a new user (ZHS) for this second study at The Queen Elizabeth Hospital, Birmingham, United Kingdom. NBI flexible cystoscopy was performed on 23 patients with known recurrences of urothelial cancer (UC) of the bladder after initial conventional WLI flexible cystoscopy with the same switchable Olympus Lucera sequential red/green/blue instrument. RESULTS: NBI detected 15 additional UCs in 8 of the 23 (35%) patients: Six of these patients had one additional UC, one had four additional UCs, and one had five additional UCs when compared with WLI, with a mean of 0.65 additional UCs per patient (standard deviation 1.30; Wilcoxon P = 0.01). When this second series is compared with our first published series, there is no statistical evidence that the excess number of UCs detected by NBI is different (Wilcoxon [unpaired] signed-rank test P = 0.74), which suggests that there is no difference between a new user and an experienced user in the application of NBI. CONCLUSIONS: We and others continue to demonstrate a significantly improved detection rate of bladder UCs with NBI cystoscopy when compared with conventional WLI cystoscopy, even for new users previously unfamiliar with this technology.

Is human papillomavirus viral load a clinically useful predictive marker? A longitudinal study.

BACKGROUND: It has been suggested that in women who test positive for high-risk human papillomavirus (HPV) types, viral load can distinguish women who are at increased risk of cervical neoplasia from those who are not. METHODS: Quantitative PCR (qPCR) was used to measure HPV copy number in serial samples taken from 60 and 58 young women previously found to have incident cervical HPV16 or HPV18 infections, respectively, using GP5+/GP6+ primers; women provided at least three samples for qPCR testing, at least one of which was positive. RESULTS: A 10-fold increase in HPV16 or HPV18 copy number was associated with a modestly increased risk of acquiring a cytologic abnormality [HPV16: hazards ratio, 1.76 (95% confidence interval, 1.38-2.25); HPV18: hazards ratio, 1.59 (95% confidence interval, 1.25-2.03)]. However, in most women, copy number increased during follow-up, before decreasing again. In women with a HPV16 infection, the median copy number per 1,000 cells was 7.7 in their first qPCR HPV-positive sample, 1,237 in the sample yielding the maximum copy number, and 7.8 in their last qPCR HPV-positive sample; corresponding copy numbers for women with HPV18 infection were 2.3, 87, and 2.4. Maximum HPV16 and HPV18 copy number did not differ significantly between women who acquired an incident cervical cytologic abnormality and those who did not. CONCLUSION: Whereas large relative increases in copy number are associated with an increased risk of abnormality, a single measurement of viral load made at an indeterminate point during the natural history of HPV infection does not reliably predict the risk of acquiring cervical neoplasia. Therefore, a single measure of HPV viral load cannot be considered a clinically useful biomarker.

Mechanisms of recurrence of Ta/T1 bladder cancer.

INTRODUCTION: Bladder cancer recurrence occurs via four mechanisms - incomplete resection, tumour cell re-implantation, growth of microscopic tumours, and new tumour formation. The first two mechanisms are influenced by clinicians before and immediately after resection; the remaining mechanisms have the potential to be influenced by chemopreventive agents. However, the relative importance and timing of these mechanisms is currently unknown. Our objective was to postulate the incidence and timing of these mechanisms by investigating the location of bladder cancer recurrences over time. PATIENTS AND METHODS: The topographical locations of tumours and their recurrences were analysed retrospectively for 169 patients newly-diagnosed with Ta/T1 bladder cancer, with median follow-up of 33.8 months. Tumours were assigned to one or more of six bladder sectors, and time to recurrence and location of recurrences were recorded. RESULTS: Median time to first tumour recurrence was 40 months. Median times between subsequent recurrences were 6.6, 7.9, 8.0 and 6.6 months for recurrences 1 to 2, 2 to 3, 3 to 4, and 4 to 5, respectively. The risk of first tumour recurrence in any given bladder sector increased by nearly 4-fold if the primary tumour was resected from that sector (P < 0.001); this association was not significant for subsequent recurrences. The proportion of tumour recurrences in multiple bladder sectors increased from 13% for the first recurrence to 100% for recurrence seven onwards. CONCLUSIONS: First tumour recurrence appears different to subsequent recurrences; incomplete resection and tumour cell reimplantation may dominate at this time-point. Only later does genuine new tumour formation appear to increase in importance. This has important implications for clinical trials, especially those involving chemopreventive agents.

Disruption of the E2 gene is a common and early event in the natural history of cervical human papillomavirus infection: a longitudinal cohort study.

Integration of high-risk human papillomavirus (HPV) types into the host-cell genome disrupts the HPV regulatory E2 protein, resulting in a loss of negative feedback control of viral oncogene expression; this disruption has been considered a critical event in the pathogenesis of cervical neoplasia, and a potential biomarker of progressive disease. However, using serial samples taken from a cohort of young women who were recruited soon after they first had sexual intercourse, we show that disruption of the E2 gene is a common and early event in the natural history of incident cervical HPV infections. The E2 gene was significantly more likely to be disrupted in women who tested positive for HPV18 in their baseline sample than in those who tested positive for HPV16 [26% versus 58%; relative risk, 2.26; 95% confidence interval (CI), 1.38-3.71; chi(2), 9.23; 1 degree of freedom (df); P = 0.002]. Among women with an intact E2 gene in their baseline sample, the median time to first detection of E2 disruption was also shorter for those who tested positive for HPV18 than HPV16 (5.7 versus 10.9 months; hazards ratio, 1.93; 95% CI, 0.84-4.44; chi(2), 2.49; 1 df; P = 0.11). This tendency for HPV18 to integrate early, coupled with the substantial reduction in viral load in HPV18-positive samples in which E2 is disrupted, may explain why HPV18-associated disease is often reported to be characterized by minor cytologic changes, which underestimate the severity of the underlying histologic abnormality.

Proximity of first intercourse to menarche and the risk of human papillomavirus infection: a longitudinal study.

Cross-sectional studies have suggested that compared with women who delay the start of their sexual career, those who first have intercourse soon after menarche are more susceptible to cervical human papillomavirus (HPV) infection and thus have a greater risk of cervical neoplasia. We describe, using longitudinal observations, how the risk of infection with HPV varies with the interval between menarche and first intercourse in 474 women aged 15-19 recruited within 12 months of first intercourse and before the acquisition of a second sexual partner. One hundred forty-five women became HPV-positive; the cumulative risk of HPV infection 3 years after first intercourse was 45.0% (95% CI = 37.9-51.2). In univariate analyses, the hazards ratio (HR) of HPV infection increased significantly with age at first intercourse (HR = 1.212 per year; 95% CI = 1.050-1.398), partner age (HR = 1.084 per year; 95% CI = 1.045-1.125) and when women reported a sexually experienced partner (HR = 2.794; 95% CI = 1.804-4.326); the interval between menarche and first intercourse was a significant predictor of infection, with an increase in the HR of 12.9% for every year of increase in this interval (95% CI = 2.1%-24.9%). In a multivariate analysis, compared with women who first had intercourse within 3 years of menarche, those who postponed first intercourse beyond this time had a greater risk of infection (HR = 1.581; 95% CI = 1.113-2.245) after controlling for age and sexual experience of partner.