A randomised, controlled, crossover study to investigate the safety and response of 1R,2S-methoxamine hydrochloride (NRL001) on anal function in healthy volunteers.

AIMS: This study aimed to assess the dose and volume effects of suppository preparations and safety of NRL001 (one of four possible stereoisomers of methoxamine hydrochloride) on anal sphincter tone using rectal suppositories in healthy adult volunteers. METHODS: This was a Phase I, single-centre, randomised, double-blind, three-way crossover study during which subjects received three single doses of 1 g rectal suppositories (containing 5 or 10 mg NRL001 or matching placebo) or 2 g rectal suppositories (containing 10 or 15 mg NRL001 or matching placebo) on three separate dosing days. The outcome measures were mean anal resting pressure (MARP) variables (monitored continuously for 20-30 min before and up to 6 h after dosing), pharmacokinetics (PK) and safety assessments. RESULTS: Twenty-six subjects were dosed with study medication. Two subjects were withdrawn prematurely and were not included in the main analysis. There was a dose-dependent increase in anal sphincter tone (MARP) when comparing the 5 and 10 mg doses of NRL001; however, the 15 mg dose did not have a significantly greater effect than the 10 mg dose. Suppository size (1 or 2 g) did not appear to have an effect on sphincter tone. There was no evidence against dose proportionality for the PK variables, but the mean maximum plasma concentration (Cmax ) for the 1 g suppository group was higher than for the 2 g group. Twenty-one adverse events were reported in 8 (30.8%) subjects. A dose dependent decrease in heart rate was noted; however, there were no adverse events reported that were related to this reduction in heart rate. CONCLUSIONS: The increase in anal sphincter tone supports the potential therapeutic use of NRL001 in treating faecal incontinence, with further studies in patients required. NRL001 was well tolerated in single doses of up to 15 mg.

Libertas: rationale and study design of a multicentre, Phase II, double-blind, randomised, placebo-controlled investigation to evaluate the efficacy, safety and tolerability of locally applied NRL001 in patients with faecal incontinence.

AIMS: Faecal incontinence affects up to 8% of adults. Associated social isolation and subsequent depression can have devastating effects on quality of life (QoL). Faecal incontinence is an underreported health problem as the social isolation and stigma that patients experience makes it difficult for sufferers to discuss their condition with a physician. There have been few well-designed, placebo-controlled clinical trials of treatment for faecal incontinence and little clinical evidence is available to inform the most appropriate management strategies. Libertas, a robustly designed study will investigate the efficacy and safety of NRL001 (1R,2S-methoxamine), an α1 -adrenoceptor agonist, in the treatment of faecal incontinence. METHODS: Libertas is a multicentre, Phase II, double-blind, randomised, placebo-controlled, parallel group study. Patient recruitment took place across 55 study centres in Europe. Patients suffering with faecal incontinence were randomised into four groups (approximately 110 each) to receive once daily self-administered doses of NRL001 (5, 7.5 or 10 mg or placebo in a suppository formulation) for 8 weeks. The primary objective of Libertas is to assess the impact of once daily administration of NRL001 on the severity and frequency of incontinence episodes as assessed by the Wexner score at 4 weeks, compared with placebo. Secondary outcomes include measures of efficacy of NRL001 compared with placebo following 8 weeks treatment; safety and tolerability; evaluation of plasma pharmacokinetics; establishment of any pharmacokinetic/pharmacodynamic relationship to adverse events; dose-response relationship; the efficacy of NRL001 therapy at 4 and 8 weeks assessed by the Vaizey score; and QoL using the Faecal Incontinence Quality of Life and the EQ-5D-5L Healthcare Questionnaires following 4 and 8 weeks NRL001 therapy. Overall patient satisfaction with the treatment will also be evaluated. DISCUSSION: This is the first randomised controlled study to investigate the efficacy and safety of a selective α1 -adrenoceptor agonist for the treatment of faecal incontinence. Furthermore, this is the first time the impact of NRL001 on assessments of QoL, health outcomes and patient satisfaction will be assessed. Innovative strategies were developed to meet the challenge of recruiting patients for this study, for example, media advertising, posters and mailshots as allowed by each study centre.

Nottingham trial of faecal occult blood testing for colorectal cancer: a 20-year follow-up.

BACKGROUND: Three large randomised trials have shown that screening for colorectal cancer (CRC) using the faecal occult blood test (FOBt) can reduce the mortality from this disease. The largest of these trials, conducted in Nottingham since 1981, randomised 152,850 individuals between the ages of 45 and 74 years to an intervention arm receiving biennial Haemoccult (FOB) test kit or to a control arm. In 2006, the National Bowel Cancer Screening Programme was launched in England using the FOBt, with the expectation that it will reduce CRC mortality. AIMS: To compare the CRC mortality and incidence in the intervention arm with the control arm after long-term follow-up. METHODS: The 152,850 randomised individuals were followed up through local health records and central flagging (Office for National Statistics). RESULTS: At a median follow-up of 19.5 years there was a 13% reduction in CRC mortality (95% CI 3% to 22%) in the intervention arm despite an uptake at first invitation of approximately 57%. The CRC mortality reduction in those accepting the first screening test, adjusted for the rate of non-compliers, was 18%. There was no significant difference in mortality from causes other than CRC between the intervention and control arms. Despite removing 615 adenomas >10 mm in size from the intervention arm, there was no significant difference in CRC incidence between the two arms. CONCLUSIONS: Although the reduction in CRC mortality was sustained, further follow-up of the screened population has not shown a significant reduction in the CRC incidence. Moreover, despite the removal of many large adenomas there was no reduction in the incidence of invasive cancer which was independent of sex and site of the tumour.

Analysis of deaths occurring within the Nottingham trial of faecal occult blood screening for colorectal cancer.

OBJECTIVE: To report the causes of, and ages at, death of subjects in an English colorectal cancer screening trial. DESIGN AND SETTING: Analysis of 78 708 deaths occurring between 1981 and 2008, within the Nottingham randomised controlled trial of biennial faecal occult blood testing. MAIN OUTCOME MEASURES: Cause of death, age at death by sex and by cause. RESULTS: Significantly more subjects died from verified colorectal cancer in the trial's control group than in the intervention group (3.2% vs 2.9%). For no other major cause of death was the difference in proportion across the two groups statistically significant. Age at death was lower for cancer than for other principal causes, except for ischaemic heart disease among women. However, mean age at death was higher for colorectal cancer than for other cancers, except for prostate cancer among men. Increasing levels of material deprivation significantly lowered the expected ages at death, independently of cause. For both men and women, the mean age at death from all causes for screening participants was higher than that of controls and non-participants. Mean deprivation was lowest among participants. Of those participating in screening, and dying from colorectal cancer, subjects receiving negative test results lived significantly longer than those who received positive test results. However, if dying from other causes, they died at an earlier age. CONCLUSIONS: The age at death from colorectal cancer is higher than that of most other cancers. Those accepting a screening invitation live longer than non-participants. In part, this difference is explained by relative deprivation. Among screening participants, the receipt of a positive, as opposed to a negative, test result is associated with a later age at death.

Population screening for colorectal cancer: the implications of an ageing population.

Population screening for colorectal cancer (CRC) has recently commenced in the United Kingdom supported by the evidence of a number of randomised trials and pilot studies. Certain factors are known to influence screening cost-effectiveness (e.g. compliance), but it remains unclear whether an ageing population (i.e. demographic change) might also have an effect. The aim of this study was to simulate a population-based screening setting using a Markov model and assess the effect of increasing life expectancy on CRC screening cost-effectiveness. A Markov model was constructed that aimed, using a cohort simulation, to estimate the cost-effectiveness of CRC screening in an England and Wales population for two timescales: 2003 (early cohort) and 2033 (late cohort). Four model outcomes were calculated; screened and non-screened cohorts in 2003 and 2033. The screened cohort of men and women aged 60 years were offered biennial unhydrated faecal occult blood testing until the age of 69 years. Life expectancy was assumed to increase by 2.5 years per decade. There were 407 552 fewer people entering the model in the 2033 model due to a lower birth cohort, and population screening saw 30 345 fewer CRC-related deaths over the 50 years of the model. Screening the 2033 cohort cost pound 96 million with cost savings of pound 43 million in terms of detection and treatment and pound 28 million in palliative care costs. After 30 years of follow-up, the cost per life year saved was pound 1544. An identical screening programme in an early cohort (2003) saw a cost per life year saved of pound 1651. Population screening for CRC is costly but enables cost savings in certain areas and a considerable reduction in mortality from CRC. This Markov simulation suggests that the cost-effectiveness of population screening for CRC in the United Kingdom may actually be improved by rising life expectancies.

Psychological and colonic factors in painful diverticulosis.

BACKGROUND: The importance of psychological factors in symptom expression in diverticulosis is unknown. This follow-up study assessed the relative importance of colonic and psychological factors in symptom expression. METHODS: Patients with barium enema-proven diverticula were sent a bowel symptom questionnaire in 1999 and again in 2006 with additional psychological questionnaires included. RESULTS: Some 170 of 261 initial responders were eligible for follow-up and 124 (72.9 per cent) provided complete replies. Forty-two (33.9 per cent) of 124 respondents experienced recurrent abdominal pain a median of 3.5 (interquartile range (i.q.r.) 2.00-9.25) days per month, with a median duration of 1 (i.q.r. 0.7-2) h. Multivariable analysis identified a history of acute diverticulitis (odds ratio 3.98; P = 0.010) and a raised score on the Hospital Anxiety and Depression Scale (odds ratio 2.53; P = 0.030) as the best predictors of recurrent pain. CONCLUSION: Psychological and colonic factors are important in symptom expression in diverticulosis.

Risk-stratified intensive follow up for treated colorectal cancer - realistic and cost saving?

OBJECTIVE: Intensive follow-up post surgery for colorectal cancer (CRC) is thought to improve long-term survival principally through the earlier detection of recurrent disease. This paper aims to calculate the additional resource and cost implications of intensive follow up post-CRC resection, examine the possibility of risk-stratifying this follow up to those at highest risk of recurrence and investigating the impact that population screening might have on the future cost and outcomes of follow up. METHOD: Two follow-up regimens were constructed: the 'standard' follow-up protocol used the principles of the British Society of Gastroenterology (BSG) guidelines whilst the 'intensive' follow-up protocol used the most intensive arm of the follow up after colorectal surgery (FACS) trial. Using ONS data, the number of CRC diagnosed in a given year was calculated for 2003 and projected for 2016 based on the population of England and Wales. The resource requirements and costs of follow up over a 5-year period were then calculated for the two time periods. Risk stratifying entry to follow up and the introduction of population CRC screening were then considered. RESULTS: For the 2003 cohort, an intensive follow-up program would detect 853 additional resectable recurrences over 5 years with 795 fewer subjects requiring palliative care. An additional 26 302 outpatient appointments, 181 352 CEA tests and 79 695 CT scans over 5 years would be required to achieve this. The cost of investigating subjects who would never develop detectable recurrences was pound15.6 million. The cost per additional resectable recurrence was pound18 077, a figure also found for a nonscreened population in 2016. An identical intensive follow-up policy with biennial FOBT screening in 2016 saw the cost per additional resectable recurrence rise to pound36 255. CONCLUSION: Intensive follow up will detect considerably more resectable recurrences but at considerable cost and it is unclear if such follow up will be achievable in an already over-stretched NHS. If population-based CRC screening increases the number of Dukes A cancers this may offer the possibility of risk-stratifying future follow up to those at highest risk of recurrence; minimizing tests on those who will never have recurrent disease and better utilizing our scarce resources.

Chemoradiotherapy with Brachytherapy or Electron Therapy Boost for Locally Advanced Squamous Cell Carcinoma of the Anus-Reducing the Colostomy Rate.

PURPOSE: The aim of this study is to determine overall survival, disease-specific survival and stoma-free survival after treatment of squamous cell carcinoma of the anus with chemoradiotherapy followed by brachytherapy or electron boost in a recent cohort of patients. METHODS: Fifty-two patients (median age 62 years) were treated with radical chemoradiotherapy (mitomycin C, infusional 5-fluorouracil concurrently with conformal radical radiotherapy 45 Gy in 25 fractions over 5 weeks) followed by a radiotherapy boost between 1 December 2000 and 30 April 2011. Follow-up was to 30 November 2014. Thirty-six patients received a boost (15-20 Gy) over 2 days with 192Ir needle brachytherapy for anal canal tumours, and 16 patients received electron beam therapy (20 Gy in 10 fractions in 2 weeks) for anal margin tumours. A defunctioning stoma was only created prior to chemoradiotherapy for fistula or severe anal pain. RESULTS: The overall survival for the 36 patients treated with chemoradiotherapy followed by brachytherapy was 75 % (95 % CI, 61-89) at 5 years, the disease-specific survival was 91 % (95 % CI, 81-101 %), and the stoma-free survival was 97 % (95 % CI, 91-103 %) all at 5 years. For the 16 patients treated with an electron boost for anal margin tumours, the 5-year overall survival, disease-specific survival and stoma-free survival were 68 % (95 % CI, 44-92 %), 78 % (95 % CI, 56-100 %) and 80 % (95 % CI, 60-100 %), respectively. CONCLUSIONS: A very low stoma formation rate can be obtained with radical chemoradiotherapy followed by a brachytherapy boost for squamous cell carcinoma of the anal canal but not with an electron boost for anal margin tumours.

Vaccine-induced apoptosis: a novel clinical trial end point?

The functional end point of immunotherapy is to induce tumor regression. Because immune effector mechanisms usually result in apoptosis, the aim of this study was to determine whether measurement of tumor apoptosis ex vivo is a good end point to evaluate the efficacy of cancer vaccines. A prototype vaccine, 105AD7, was administered to colorectal cancer patients before resection of their primary tumors. There was a significant increase in apoptosis of tumor cells within immunized patients compared with control patients as assessed by immunohistochemistry (P = 0.005; n = 16) or by flow cytometry (P = 0.003; n = 34). Preoperative immunization and measurement of tumor cell apoptosis may be a valuable clinical end point for evaluation of new vaccine and other biological approaches.

Radiation induced MMP expression from rectal cancer is short lived but contributes to in vitro invasion.

AIMS: Matrix metalloproteinase (MMP) activity is increased after radiation. The aims of this study were to assess the time course of this increase and its effects on malignant cell invasion. METHODS: Colorectal cancer (HCT 116, LoVo, C 170 HM 2, CaCO-2), fibroblast (46-BR.IGI, CCD-18 Co) and fibrosarcoma (HT1080) cell lines were irradiated at 4 gray (4 Gy) and matrix metalloproteinase gene and protein expression examined over a 96 h period by real time polymerase chain reaction and gelatin zymography. Invasion was assessed on Matrigel. Human rectal tumour MMP expression was compared before and after long course radiotherapy. RESULTS: Radiation increased MMP gene expression of tumour cell lines, and resulted in increased MMP protein activity in the HT1080 line. HT1080 and HCT 116 in monoculture and LoVo in co-culture were more invasive after radiation at 48 h in vitro, but long course radiotherapy did not result in a consistent increase in MMP expression from human rectal tumour biopsies. CONCLUSIONS: Radiation results in increased MMP expression for a limited time period. This results in an early increase in cell line invasion. Further clinical research is required to clarify if MMP inhibition given perioperatively following radiotherapy decreases local recurrence rates.

A neoadjuvant clinical trial in colorectal cancer patients of the human anti-idiotypic antibody 105AD7, which mimics CD55.

Thirty-five patients received 105AD7 human anti-idiotype vaccination prior to surgery for colorectal carcinoma. Patients were immunized before and also received one to two immunizations after surgical resection of their colorectal cancer. The vaccine was well tolerated with no associated toxicity. Lymphocytic infiltration within the resected tumors was quantified by immunohistochemistry and image analysis. Enhanced infiltration of helper T cells (CD4) and natural killer (NK) cells (CD56) were observed in the tumors from immunized patients when compared with tumors from stage, grade, site, age, and sex matched unimmunized patients. NK activity was increased in the blood, peaking 7-10 days post immunization and then dropping rapidly and correlating with NK extravasation within the tumor. Comparison of the amino acid sequences of 105AD7 anti-idiotype and the antigen it mimics, CD55, has predicted that patients with HLA-DR1, HLA-DR3, and HLA-DR7 haplotypes should show helper T cell responses following 105AD7 vaccination. Eighty-three percent of patients expressing these haplotypes responded to 105AD7, whereas 88% of patients who failed to express these haplotypes were nonresponders. With a median follow-up of 4 years (range, 2.5-6 years) 65% of patients remained disease free. This trial shows that 105AD7 stimulates antitumor inflammatory responses allowing extravasation within tumor deposits of both helper T cells and NK cells. This represents a way of evaluating immune responses in patients both within the blood and at the tumor site. The study confirms that immunization with a human anti-idiotypic antibody results in immune responses in 83% of patients with a permissive haplotype.

Effect of preoperative radiotherapy on matrilysin gene expression in rectal cancer.

Matrilysin, a member of matrix metalloproteinase family, is believed to play a significant role in the growth and proliferation of colon cancer cells. Overexpression of the matrilysin gene has been shown to correlate with Dukes' stage and increased metastatic potential in colorectal cancer. The aim of this study was to evaluate the effect of preoperative high-dose radiotherapy (25 Gy in five fractions over 5 days) on matrilysin (MMP-7) gene expression, in patients with resectable rectal cancer, by a quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Biopsy samples of tumour (n=30) and distant normal mucosa (n=12) from 15 patients were obtained pre- and post-radiotherapy. Messenger (m)RNA was extracted from all of the tissue samples and reverse transcribed to double-stranded cDNA. Quantitative RT-PCR was performed to study the effect of preoperative radiotherapy on matrilysin gene expression in both the tumour and normal mucosal specimens. Matrilysin mRNA values were expressed relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for each sample. In 14 out of 15 cases, matrilysin mRNA was detected in the cancerous tissue. Although all six normal mucosal specimens expressed matrilysin mRNA, the levels were approximately 10-fold lower compared with those seen in the paired tumour samples. Preoperative radiotherapy led to a significant 6- to 7-fold increase (P=0.001) in the expression of matrilysin mRNA in rectal cancer tissue. In contrast, there was no significant change in the matrilysin mRNA expression of normal mucosal specimens post-radiotherapy. Preoperative high-dose radiotherapy upregulates matrilysin gene expression in rectal cancer. Matrilysin inhibition may be a useful preventive or therapeutic adjunct to radiotherapy in rectal cancer.

Pharmacological characterization of neurogenic responses of the sheep isolated internal anal sphincter.

The aim of the study was to establish the nature of the neurogenic responses of the sheep isolated anal sphincter. Isolated strips of sheep internal anal sphincter develop intrinsic contractile tone following the application of stretch tension. On transmural stimulation (1 - 20 Hz, 10 V pulse strength, 0.5 ms pulse width, 1 s every 180 s) transient relaxations were observed. The amplitude of the relaxations were frequency-dependent reaching a maximal response at 10 - 20 Hz and were inhibited by tetrodotoxin (0.3 microM). Neither atropine (0.3 microM) nor phentolamine (1 microM) affected control responses. The nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 100 microM) and the selective inhibitor of soluble guanylyl cyclase ODQ, (1H-[1,2, 4]oxadiazolo[4,3-a]quinoxalin-1-one) (1 microM) completely inhibited the neurogenic relaxations and uncovered contractions that were abolished by 1 microM phentolamine and 0.1 microM prazosin. The effect of L-NAME, but not that of ODQ, was partially reversed by the addition of L-arginine (1 mM). Sodium nitroprusside (10 nM - 10 microM) caused concentration-dependent inhibition of myogenic tone and this effect was significantly reduced by ODQ. Calcium-free Krebs-Henseleit solution also reduced myogenic tone by 85%. Transmural electrical stimulation of the sheep isolated internal anal sphincter causes a transient relaxation of myogenic tone that appears to involve nitric oxide from non-adrenergic, non-cholinergic nerves and, to a lesser degree, noradrenaline from sympathetic nerves. The characteristics of the preparation compares well with that of human tissue and may prove to be a suitable animal based model for further studies.

Nucleolar organiser regions (AgNORS) in anal intraepithelial neoplasia and invasive anal squamous cell carcinoma.

AIM: To evaluate the usefulness of counting nucleolar organiser region associated proteins (AgNORs) in the management of anal squamous neoplasia. METHOD: Using a silver staining technique for NOR associated proteins, 32 routinely processed paraffin wax embedded sections of anal epithelium were assessed. These consisted of normal anal epithelium (n = 9), anal intraepithelial neoplasia (AIN) grades I (n = 5), and III (n = 13), and invasive squamous neoplasia of the anus (n = 5). RESULTS: The median AgNOR counts for every 100 cells are as follows: normal anal epithelium 2.15 (95% CI 1.89-3.94); AIN I 3.21 (95% CI 2.89-7.14); AIN III 4.32 (95% CI 4.00-8.10); and invasive squamous cell carcinoma of the anus 5.51 (95% CI 2.48-10.62). There were significant differences between AgNOR counts in anal cancer and normal epithelium (p < 0.05; Mann-Whitney U test)), AIN III and normal anal epithelium (p < 0.005), and AIN III and AIN I (p < 0.05). No significant differences were observed between AIN I and normal anal epithelium, anal cancer and AIN I, and anal cancer and AIN III. There was a considerable degree of overlap among the different groups. CONCLUSIONS: Despite the strong association between AgNOR values and degree of dysplasia, the variability within pathological grade may preclude the adoption of this technique on its own as a prognostic indicator. It may, however, be useful in conjunction with other markers of neoplastic growth such as c-myc oncogene amplification or overexpression as a marker of disease progression in AIN and invasive anal squamous cell cancer.

C-myc oncogene expression in anal squamous neoplasia.

AIMS: To determine the pattern of c-myc oncogene expression in anal squamous neoplasia and to determine if this could be used as a marker of disease progression. METHODS: The presence and localisation of the c-myc gene product p62 in archival specimens of anal squamous epithelium, normal and neoplastic, was examined using immunohistochemical staining with the monoclonal antibody Myc1-6E10. Ten normal and epithelia, 10 anal intraepithelial neoplasia (AIN) III, and 31 anal squamous cancers were examined. RESULTS: There was a noticeable difference between the staining characteristics of invasive tumours, normal anal epithelium, and AIN III. Intense, diffuse, mixed nuclear and cytoplasmic (n = 14) and exclusively nuclear (n = 8) staining in 22 of 31 (71%) of invasive anal tumours was observed. All positively staining tumours were well differentiated histologically, while the negatively staining nine of 31 (29%) were poorly differentiated (n = 7) and moderately well differentiated (n = 2). In six positively staining tumour sections adjacent areas of AIN III and non-dysplastic anal epithelium had staining characteristics similar to those of the invasive component. Staining in both normal anal epithelium (4/10) and AIN III specimens obtained from patients without a history of invasive disease (8/10) was less intense, focal in distribution, and exclusively nuclear. No difference in staining characteristics could be detected in these two groups. CONCLUSIONS: The results of this study suggest that c-myc oncogene expression is implicated in the pathogenesis of anal squamous neoplasia, and that immunohistochemical staining for c-myc protein may be helpful in identifying those AIN III lesions most likely to progress to invasive tumours.

Immunohistochemical analysis of p53 expression in anal squamous neoplasia.

AIMS: To determine the pattern of expression of the p53 tumour suppressor gene product in anal squamous neoplasia, and to determine if this could be used as a marker of disease progression. The association between p53 expression and human papillomavirus (HPV) 16 DNA status of the anal lesions was also investigated. METHODS: The presence and localisation of the p53 protein in formalin fixed, paraffin wax embedded specimens of anal squamous epithelium (normal and neoplastic) was examined using immunohistochemical staining with a panel of two monoclonal antibodies (DO-1, DO-7) and one polyclonal antibody (CM-1). Thirty nine normal anal epithelia, 14 anal intraepithelial neoplasia (AIN) grade 1, seven AIN 2, and 20 AIN 3 specimens were obtained from patients without demonstrable invasive disease; twelve AIN 3 specimens adjacent to invasive disease and 34 anal squamous cancers were also examined. Genomic DNA from all 126 specimens was extracted and analysed for HPV 16 DNA using the polymerase chain reaction (PCR). RESULTS: Nuclear p53 was strongly expressed in 67% (23/34) of invasive anal squamous tumours, 75% (9/12) of AIN 3 specimens adjacent to invasive disease, and in 60% (12/20) of AIN 3 specimens obtained from patients without demonstrable invasive disease. Two of the patients in the latter group with positively staining specimens subsequently developed invasive tumours which had staining characteristics similar to those of the AIN 3 specimens. p53 protein was expressed in very low concentrations in low grade AIN and not at all in normal anal squamous epithelium. In those specimens which stained positively for p53, HPV 16 DNA sequences were detected in 69.5% (16/23) of invasive disease, 77.7% (7/9) of AIN 3 adjacent to invasive disease, 75% (9/12) of AIN 3 obtained from patients without demonstrable invasive disease, 33.3% (2/6) of AIN 2, and in 40% (2/5) of AIN 1. There was no significant correlation between p53 immunostaining and HPV 16 DNA status (p < 0.05). CONCLUSIONS: Aberrant expression of the p53 gene product is probably involved in the pathogenesis of anal squamous neoplasia. Long term follow up studies of all patients with AIN are required to determine if this could be used as a marker of likely disease progression from high grade AIN to invasive disease. There does not seem to be an association between the presence or absence of HPV 16 DNA sequences and mutant p53 proteins in anal squamous neoplasia.

DNA hybridisation of routinely processed tissue for detecting HPV DNA in anal squamous cell carcinomas over 40 years.

To study possible changes in the incidence of human papillomavirus (HPV) associated anal squamous cell carcinomas (SCC) a simple, rapid, and sensitive technique (alkaline hydrolysis) to permit DNA hybridisation from formalin fixed, paraffin wax embedded tissue was developed. The sensitivity and specificity of the technique were established by comparison with Southern blot analysis and in situ hybridisation on the same tissue specimens. Ninety tissue specimens in a single analysis were examined using this technique. Alkaline hydrolysis was applied to fixed tissue samples which showed a two-fold increase over the past 10 years in the percentage of anal cancers containing HPV type 16 DNA when compared with the previous 30 years using 207 cases of anal cancer collected over a 40 year period. This method has several advantages over the polymerase chain reaction as it is simple, relatively inexpensive, and may be widely applied to the detection and quantification of DNA sequences, including cellular oncogenes.