Significant Neoplasia Research Articles

Factors affecting faecal immunochemical test positive rates: demographic, pathological, behavioural and environmental variables

Positive rates in faecal immunochemical test (FIT)-based colorectal cancer screening programmes vary, suggesting that differences between programmes may affect test results. We examined whether demographic, pathological, behavioural, and environmental factors affected haemoglobin concentration and positive rates where samples are mailed. A retrospective cohort study; 34,298 collection devices were sent, over five years, to screening invitees (median age 60.6). Participant demographics, temperature on sample postage day, and previous screening were recorded. Outcomes from colonoscopy performed within a year following FIT were collected. Multivariate logistic regression identified significant predictors of test positivity. Higher positive rate was independently associated with male gender, older age, lower socioeconomic status, and distally located neoplasia, and negatively associated with previous screening (p < 0.05). Older males had higher faecal haemoglobin concentrations and were less likely to have a false positive result at colonoscopy (p < 0.05). High temperature on the sample postage day was associated with reduced haemoglobin concentration and positivity rate (26-35℃: Odds ratio 0.78, 95% confidence interval 0.66-0.93), but was not associated with missed significant neoplasia at colonoscopy (p > 0.05). Haemoglobin concentrations, and therefore FIT positivity, were affected by factors that vary between screening programmes. Participant demographics and high temperature at postage had significant effects. The impact of temperature could be reduced by seasonal scheduling of invitations. The importance of screening, and following up positive test results, particularly in older males, should be promoted.

Urinary PGE-M in colorectal cancer: predicting more than risk?

Progress in cancer chemoprevention has been hindered by a lack of validated biomarkers of risk and interventive response. The identification of accurate, reliable, and easily measurable risk and response biomarkers within the field of cancer prevention could dramatically alter our approach to the disease. Colorectal cancer is associated with substantial morbidity and a limited 5-year survival rate for late-stage disease. The identification of biomarkers to predict (i) those most at risk of clinically significant colorectal neoplasia in conjunction with or building upon current risk models and/or (ii) those most likely to respond to potential colorectal chemopreventive agents, such as aspirin and NSAIDs, would significantly advance colorectal cancer risk management. Urinary PGE-M is an established indicator of systemic prostaglandin E2 production and has previously been demonstrated to predict risk of advanced colorectal neoplasia in a handful of studies. In the July 2014 issue, Bezawada and colleagues confirmed those earlier risk associations and demonstrated that PGE-M can also predict responsiveness to aspirin/NSAIDs in a small subset of women undergoing lower endoscopy in the Nurse's Health Study. PGE-M has the potential to define subsets of the population that may derive greater chemopreventive benefit from NSAIDs, as well as the potential to optimize the use of expensive and/or invasive screening tests. Additional larger and more diverse prospective studies meeting the criteria for phase IV biomarker studies are needed to advance the development of PGE-M as a noninvasive biomarker of both risk and chemopreventive response in populations at risk for colorectal cancer.

Abstract 882: Non-invasive diagnostic testing for lung cancer in a prospective cohort

Abstract Monoclonal antibody Adnab-9 recognizes the p87 antigen, a constituent of putatively activated pulmonary type 2 pneumocytes. p87 shed into sputum and swallowed may be a potential tumor marker detectable by the Adnab-9 stool test. We describe prospectively collected stool and colonic effluent ELISA results in a cohort of urban patients enrolled in a colorectal cancer screening study. Methods: Prospectively, 1,132 American patients in Metropolitan Detroit submitted stool in a CRC prospective screening study from 1996 to 2013. Eleven patients free of any significant colorectal neoplasia or other cancer, but who had died from non-cancer-related causes, served as controls. Patients and controls were approximately 50-80 years old and the male to female ratios were similar (30:1 and 10:1). Sixty-seven percent of patients and 45 % of controls were African American. Results: 64 patients of 1,132 contracted lung cancer during the 17 year period of follow-up (5.7%). Of these 64 patients, 34 (52%) had stool or effluent Adnab-9 binding tests on retrospective review. We found that over half the patients ultimately diagnosed with lung cancer had either stool/effluent ELISA or Western blot positivity for Adnab-9 compared to only 1 of 11 controls (OR = 11.3, CI 1.3-97.9, p=0.014). The median time from stool submission to diagnosis of lung cancer was 4.4 (0.7-10.3) years. Lung Cancer Tumor types were adenocarcinoma in 23%; squamous in 33%; other in 20%; large cell cancer in 9%; small cell cancer in 8%; and unknown in 7%. At the last time of follow-up, 70% of patients subsequently diagnosed with lung cancer were deceased. Median survival time was 2.1 years from the time of clinical diagnosis. Conclusion: Adnab-9 sensitivity in this cohort was 52% with specificity of 90.9% for lung cancer and could lead to the diagnosis of lung cancer 4.4 years before clinical diagnosis was made. This supports the notion that accessible, early non-invasive testing combined with effective follow-up studies may lead to earlier diagnosis and treatment of lung cancer, the World's most frequent cancer. Citation Format: Martin Tobi, Joe Sayre, MaryAnn Rambus, Fadi Antaki, Ann Schwartz, Michael J. Lawson. Non-invasive diagnostic testing for lung cancer in a prospective cohort. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 882. doi:10.1158/1538-7445.AM2014-882

Flexible sigmoidoscopy following a positive faecal occult blood test within a bowel screening programme may reduce the detection of neoplasia

Colorectal cancer screening using the faecal occult blood test (FOBt) detects a disproportionate number of left-sided tumours. This study aims to examine the theoretical impact on neoplasia detection rates of a sigmoidoscopy-first protocol in FOBt-positive patients undergoing colonoscopy. From retrieved endoscopy/pathology reports, pathology up to and including the splenic flexure was assumed detectable by sigmoidoscopy. High-risk polyps prompting subsequent colonoscopy were classed as three or more polyps, one polyp of ≥ 1 cm, villous or tubulovillous components or the presence of high-grade dysplasia. Between April 2009 and April 2011, 4631 patients underwent colonoscopy as a result of a positive FOBt in Greater Glasgow and Clyde. Cancer was detected in 398 (9%) and adenomas were detected in 1985 (47%) of which 1323 (67%) were deemed significant according to British Society of Gastroenterology guidelines. Applying the flexible sigmoidoscopy-first model, cancer would have been detected in 329 (8%) patients and adenomas in 1640 (39%), of which 1140 (70%) would have been significant. In total, 1546 (37%) patients would have required subsequent colonoscopy, following which 21 patients would have a new diagnosis of cancer. The positive predictive values (PPVs) for neoplasia (47 vs 57%, P < 0.001), significant neoplasia (35 vs 41%, P < 0.001) and cancer (8 vs 9%, P = 0.007) were all lower in the sigmoidoscopy-first model. A significant reduction in the detection of both adenomas and cancers would be seen if the sigmoidoscopy-first protocol were to be used following a positive FOBt. Furthermore, a significant proportion of patients would be subjected to two procedures, with considerable implications for both the patient and cost.

Colonoscopy Following Nonoperative Management of Uncomplicated Diverticulitis May Not Be Warranted

Following the nonoperative management of acute diverticulitis, guidelines recommend routine follow-up colonoscopy; however, evidence to support this recommendation are lacking. This study aims to determine the diagnostic yield of endoscopy for clinically significant neoplasia following the successful nonoperative management of acute diverticulitis. This study is a retrospective review. This study was conducted in a large urban health region. Adult patients who were admitted with a diagnosis of acute diverticulitis confirmed by CT and who were successfully managed nonoperatively to hospital discharge were included. Patients who underwent colonoscopy within 2 years of presentation were excluded. The primary outcome measured was the incidence of clinically significant neoplasia (invasive malignancy or advanced adenoma) on follow-up endoscopy within 1 year of admission. Four hundred fifty-eight patients were selected for analysis, of which 249 patients (54%) underwent endoscopy within 1 year of admission. Seventy-seven (30.9%) patients were found to have polyps, 19 (7.6%) patients had advanced adenomas, and 4 (1.6%) patients had an invasive malignancy; 23 patients (9.2%) were found to have clinically significant neoplasia. On subgroup analysis, patients presenting with complicated diverticulitis (n = 74) had a significantly higher incidence of advanced adenoma (18.9% vs 5%, p = 0.001) and invasive malignancy (5.4% vs 0%, p = 0.007) in comparison with patients who presented with uncomplicated diverticulitis (n = 175). On multivariate analysis, patient age (OR 1.04 (1.01-1.08), p = 0.02) and the presence of abscess (OR 4.15 (1.68-10.3), p = 0.002) were identified as significant risk factors for clinically significant neoplasia. The use of retrospective data was a limitation of this study; 54% of selected patients underwent endoscopic follow-up. The incidence of clinically significant neoplasia on endoscopic follow-up after the nonoperative management of acute diverticulitis is 9.2%. Those with complicated diverticulitis are at higher risk, whereas the incidence of clinically significant neoplasia in those with uncomplicated diverticulitis is equal to the incidence in average-risk individuals. Routine diagnostic colonoscopy following the nonoperative management of acute uncomplicated diverticulitis may not be warranted.

Does acetylsalicylic acid or warfarin affect the accuracy of fecal occult blood tests?

Current guidelines for screening of colorectal cancer do not offer specific recommendations for cessation of antithrombotic agents prior to fecal occult blood test (FOBT). To asess the accuracy of FOBT in patients taking acetylsalicylic acid (ASA) or warfarin. A literature search was conducted for studies that investigated the accuracy of FOBT in patients taking ASA and warfarin. The primary outcome was the pooled relative risk (RR) for true positive FOBT for detecting significant colonic neoplasia in patients taking ASA or warfarin compared with controls. The secondary outcome was a pooled RR for true positive in guaiac FOBT (g-FOBT) compared with immunochemical FOBT (i-FOBT). Five observational studies included 759 patients taking ASA and 1652 control subjects. In patients taking ASA, pooled RR for true positive FOBT was 0.82 (95% confidence interval [CI] 0.73-0.93, P=0.0009), pooled RR for true positive g-FOBT was 0.69 (95% CI 0.60-0.79, P<0.0001), whereas pooled RR for true positive i-FOBT was 1.013 (95% CI 0.81-1.30, P=0.8182). Five observational studies included 806 patients taking warfarin and 10 338 control subjects. In patients taking warfarin, pooled RR for true positive FOBT was 1.559 (95% CI 1.349-1.801, P<0.0001). The results of our meta-analysis demonstrate that in patients taking ASA, there is a decrease in the positive predictive value (PPV) of g-FOBT but no significant difference in the PPV of i-FOBT compared with control subjects for detecting significant neoplasia. In patients taking warfarin, the PPV of FOBT was increased for detection of colorectal cancer compared with control subjects.

Mo1176 Screening the Worried Well: Symptomatic Individuals Are Less Likely to Have Significant Neoplasia At Colonoscopy Following a Positive Colorectal Cancer Screening Test

Mo1176 Screening the Worried Well: Symptomatic Individuals Are Less Likely to Have Significant Neoplasia At Colonoscopy Following a Positive Colorectal Cancer Screening Test

Will CT colonography improve colorectal cancer screening compliance?

Colorectal cancer (CRC) is the third most common cancer in men and women in the USA and is the second leading cause of cancer death. Despite the widespread publication of evidence-based, expert consensus guidelines for the screening and surveillance of CRC, overall adherence to CRC screening recommendations remains suboptimal. CT colonography (CTC) is a minimally invasive, sedation-free CRC screening modality that has demonstrated comparable ability with colonoscopy to detect clinically significant colorectal neoplasia and CRC. Barriers to widespread CTC implementation include the absence of a recommendation by the US Preventative Services Task Force and lack of Medicare coverage, concerns regarding radiation exposure and extracolonic findings, and the inherent difficulties involved in integrating CTC with current practice. Emerging data have demonstrated that CTC can improve CRC screening adherence and there is a current need for regulatory re-evaluation of CTC to facilitate additional gains in CRC scree...

Critical considerations of fecal occult blood tests for colorectal cancer

1University of Dundee, Nethergate, Dundee, DD1 4HN, Scotland, UK *Author for correspondence: r.j.c.steele@dundee.ac.uk The most commonly used strategy in colo­ rectal cancer (CRC) screening pro grams involves the use of stool tests to detect occult blood, and guaiac fecal occult blood tests (gFOBTs) are, to date, the only fecal tests shown to reduce CRC mortality in population­based randomized trials [1]. However, gFOBTs also carry disadvantages. A major concern is that interval cancers account for around 50% of cancers detected in gFOBT screened populations [2] and recent data show that gFOBTs are less sensitive in women than in men, and in both rectal and right­sided cancers when compared with left­sided disease [2,3]. In addition, the test is associated with a high false­positive rate with no neoplasia detected in around half of colonoscopies performed following a positive gFOBT [2,3]. This may be, in part, explained by the fact that gFOBTs are not specific for human hemoglobin (Hb) and are subject to possible dietary interference from, for example, red meat and high­peroxidase fruits and vegetables. As a result, it has been common practice to instruct participants to adhere to dietary restrictions ahead of sample collection, although this may act as a barrier to screening and affect participation rates [4]. However, data from a meta­analysis did not support dietary restrictions with gFOBTs, leading to recommendations that restrictions are abandoned to improve adherence rates [5]. Fecal immunochemical tests (FITs) are now available and are increasingly being used in screening programs. Unlike gFOBTs, FITs are specific for the detection of human Hb, eliminating any potential for dietary interference, and are also more specific than gFOBT for lower gastrointestinal bleeding. In addition, modern FITs generally allow a more convenient method of sample collection, and are associated with better participation rates [6]. Another major advantage of FIT is that automated versions not only eliminate interobserver variability, but also the quantitative nature of these tests allow provision of a measured fecal Hb concentration. Screening program “...the adoption of fecal immunochemical tests in colorectal cancer screening programs has potential to address participation, appropriate positivity rates and the problem of false‐negative results.” EDITORIAL

Clinicopathologic Importance of Women with Squamous Cell Carcinoma Cytology on Siriraj Liquid-Based Cervical Cytology

The purposes of this study were to determine the prevalence and predictive value to detect significant neoplasia and invasive lesions, and to evaluate the correlation between clinical and histopathology of women with squamous cell carcinoma (SCCA) on Siriraj liquid-based cervical cytology (Siriraj-LBC). The computerized database of women who underwent Siriraj-LBC at Siriraj Hospital, Mahidol University from January 2007 to December 2010 were retrieved. The hospital records of women with SCCA cytology were reviewed. The prevalence of SCCA cytology was 0.07%. A total of 86 women, mean age was 58.1 years. Sixty-one women (70.9%) were post-menopausal. Overall significant pathology and invasive gynecologic cancer were detected in 84 women (97.7%) and 71 women (82.5%), respectively. The positive predictive values for detection of significant neoplasia and invasive lesion were 97.7% and 82.6%, respectively. The cervical cancer was diagnosed in 69 women and among these 58 women were SCCA. Thirteen women (15.1%) had cervical intraepithelial neoplasia (CIN) 3 and two women (2.3%) had cervicitis. The sensitivity and specificity of colposcopy for cervical cancer detection in SCCA cytology were 83.3% and 75%, respectively. Median follow up period was 17.6 months and 64 patients were alive without cytologic abnormality. The final histopathology of SCCA cytology in our populations demonstrated a wide variety, from cervicitis to invasive cancer and the most common diagnosis was invasive cervical cancer. Colposcopy with biopsy and/or endocervical curettage and loop electrosurgical excision procedure should be undertaken to achieve histologic diagnosis.

Improvements in colorectal cancer screening programmes - quantitative immunochemical faecal occult blood testing - how to set the cut-off for a particular population

The aim of the study was to determine the optimum cut-off value of the quantitative immunochemical test (q-FIT) OC-Sensor for colorectal cancer and advanced adenomatous polyps in a particular population. 815 patients were referred for colonoscopy and were offered two q-FIT examinations at two different colonoscopy centers. The patients were classified according to the colonoscopic findings. Test sensitivity, specificity, and accuracy were statistically evaluated using one test and two tests at the levels of 50, 75, 100, 125, and 150 ng/mL of faecal hemoglobin in those patients with advanced polyps and colorectal cancer. The optimum cut-off test level for clinically significant neoplasia was determined using one test. The optimum cut-off value of q-FIT OC-Sensor for the detection of clinically significant neoplasia in our particular population was determined as 75 ng/mL using one test. This value provides an optimum proportion of 73% sensitivity (±95% CI 60.3% - 83.4%) and 90% specificity (±95% CI 86.8% - 92.8%), PPV and NPV were determined as 54.76% and 95.43% respectively. The first step in the implementation of q-FIT test in the screening program in our country is to determine the optimum cut-off level for a population, and to estimate the number of tests performed with respect to the optimum cost effectiveness and economical climate. Using one test, the optimum level of q-FIT OC-Sensor® in the Czech Republic was determined as 75 ng/mL. This study could serve as a model for further studies in other countries, where screening does not yet exist.

PWE-093 The NHS bowel cancer screening programme, Southern Hub—screening activity and outcomes

IntroductionAs part of the NHS Bowel Cancer Screening Programme (BCSP) in England, every man and woman registered with a GP, living in England and aged 60–74 years, is invited to...

OC-046 Guidelines on the endoscopic assessment of gastric polyps: is the workload justified?

IntroductionBritish Society of Gastroenterology guidelines on the management of gastric polyps1 recommend increased intensity of evaluation compared with traditional practice, including biopsy of ALL polyps, biopsy of intervening gastric mucosa...

Tu1183 Fecal Immunochemical Test Accuracy for Colorectal Cancer and Significant Neoplasia Detection in Average-Risk Population

Tu1183 Fecal Immunochemical Test Accuracy for Colorectal Cancer and Significant Neoplasia Detection in Average-Risk Population

Diagnostic performance of quantitative fecal immunochemical test and multivariate prediction model for colorectal neoplasms in asymptomatic individuals

Quantitative fecal immunochemical test (QTFIT) has the advantage of being able to describe test characteristics on a scaled rather than binary system. The aims of this study were to decide the optimal cut-off points of QTFIT and to make a multivariate prediction model for colorectal neoplasms in asymptomatic adults. We retrospectively analyzed 1085 consecutive asymptomatic individuals who completed both full colonoscopy and QTFIT at a general health checkup clinic. Advanced adenomatous polyps (AP) were defined as APs of at least 1 cm in diameter; adenomas with villous component or high-grade dysplasia; and significant neoplasia (SN) including both advanced AP and colorectal cancer. The ideal cut-off value of QTFIT was chosen based on a value that maximized the sum of both sensitivity and specificity, and clinical utility. For AP, 25 ng/ml was chosen as the optimal cut-off value and provided a sensitivity of 31% [95% confidence interval (CI): 27-36] and specificity of 79% (95% CI: 76-82). For SN, the ideal QTFIT cut-off value was 25 ng/ml, providing a sensitivity of 51% (95% CI: 39-62) and specificity of 77% (95% CI: 74-80). For colorectal cancer, the optimal cut-off point was 50 ng/ml, offering a sensitivity of 75% (95% CI: 41-93) and specificity of 86% (95% CI: 85-86). The multivariate prediction model was represented by nomogram and was validated by bootstrap method. The diagnostic performance of QTFIT for CRC is promising, although its sensitivity for AP and SN is unsatisfactory. BMI, in addition to age and sex improves the accuracy of SN screening by QTFIT.

Risk of Occult Malignancy in Morcellated Hysterectomy

Uterine morcellation is performed only when significant neoplasia is not anticipated. In this study, we aimed to determine the prevalence of unexpected pathology in a series of low-risk morcellated hysterectomies. We reviewed a series consisting of all patients undergoing hysterectomy with morcellation at a tertiary-care hospital over a 4-yr period (n=101). Patient records were reviewed to retrieve demographics, details of preoperative evaluation (Pap smear, endometrial biopsy, imaging), and surgical pathology diagnoses. The median number of blocks submitted for histology was 6. On final pathology, endometrium was detected in 99% of all cases. No endometrial, myometrial, or cervical neoplasia other than leiomyoma (numerous cases) was present in the morcellated uteri, but in 1 case an atypical trophoblastic nodule with necrosis and myometrial infiltration, suspected to represent epithelioid trophoblastic tumor, was inadvertently morcellated. From this series, the prospective risk of occult malignancy in a low-risk population undergoing morcellation is estimated at 1% (95% confidence interval, <0.01%-5.94%). A subgroup analysis of patients who participated in what we propose as a complete preoperative workup, consisting of nonconcerning Pap smear, endometrial biopsy, and ultrasound or magnetic resonance imaging, showed no significant findings on final histology. Even with a complete workup, however, morcellation of occult uterine malignancy remains a possibility. This risk should be discussed as part of informed consent before morcellation.

Prevalence and Risk of Significant Colorectal Neoplasia in Asymptomatic First-Degree Relatives of Patients With Colorectal Cancer

Prevalence and Risk of Significant Colorectal Neoplasia in Asymptomatic First-Degree Relatives of Patients With Colorectal Cancer

Anticoagulant or aspirin treatment does not affect the positive predictive value of an immunological fecal occult blood test in patients undergoing colorectal cancer screening

The immunochemical fecal occult blood test (i-FOBT) is widely used as a recommended screening strategy for colorectal cancer (CRC). A growing number of patients potentially targeted by CRC screening programs are on oral anticoagulant or chronic low-dose aspirin therapy, mainly for primary or secondary cardiovascular prophylaxis. This study aims at evaluating whether the use of these medications may impact on the diagnostic performances of i-FOBT for CRC screening. All i-FOBT-positive patients on anticoagulant or chronic low-dose aspirin therapy recorded in a regional mass screening program database were enrolled as cases. Control groups were derived from the same database and included drug-naive i-FOBT-positive patients, matched in a ratio of 1 : 2 for age (± 3 years of age), sex, date of colonoscopy, and practice site. Information about the use of medications was collected by cross-checking patients' interview before colonoscopy and data recorded in the provincial electronic registry of medical prescriptions. The positive predictive value of i-FOBT for significant neoplasia (high-risk adenoma and CRC) was calculated in the case and control groups. In a 2-year study period, 2376 patients were recorded in the regional database. Of these patients, 53 (2%) were on anticoagulation (control group of 106 patients) and 172 (6.6%) were on chronic low-dose aspirin treatment (control group of 344 patients). Significant neoplasia was detected in 15 (28.3%) patients on anticoagulants and in 37 (34.9%) corresponding controls (P=0.45). Significant neoplasia was detected in 50 (29.1%) patients on chronic low-dose aspirin and in 107 (31.1%) corresponding controls (P=0.64). The positive predictive value of i-FOBT for significant neoplasia is not affected by ongoing anticoagulant or chronic low-dose aspirin therapy. This finding suggests that there is no need to interrupt these treatments before i-FOBT for CRC screening.

The implication of initial 24-core transrectal prostate biopsy protocol on the detection of significant prostate cancer and high grade prostatic intraepithelial neoplasia

To assess the diagnostic value of an initial 24-sample transrectal ultrasound guided (TRUS) prostate biopsy protocol compared to the 10-core technique. We retrospectively reviewed the prostate biopsy database of consecutive men undergoing prostate biopsies under local anesthesia by using the 10 (Group A) and 24 (Group B) protocols. Men were stratified according to biopsy protocol and PSA levels. Exclusion criteria were age ≥ 75 years and PSA > 20 ng/mL. The Mann-Whitney U and Fisher's exact test were used for statistical analysis. Between April 2007 and August 2009, 869 men underwent TRUS prostate biopsies of which 379 were eligible for the study. Group A (10-cores) consisted of 243 (64.11%) men and group B (24-cores) included 139 (35.89%) men. The overall prostate cancer detection rate was 39.09% and 34.55% in Group A and B, respectively (p = 0.43). An overall 9.8% increase in Gleason 7 detection rate was found in Group B (p = 0.24). The high-grade prostatic intraepithelial neoplasia (HGPIN) detection rate in men with negative initial biopsies was 15.54% and 35.55% in Group A and B, respectively (p < 0.001). In patients with PSA < 10 ng/mL, the 24-core technique increased Gleason 7 detection rate by 13.4% (p = 0.16) and HGPIN by 23.4% (p = 0.0008), compared to the 10 core technique. The 24-core technique increased the concordance between needle biopsy and prostatectomy specimen compared to 10-core technique (p < 0.002). The initial 24-core prostate biopsy protocol did not show any benefit in the detection of prostate cancer compared to the 10-core technique. However, it improved the HGPIN detection and the correlation between biopsy results and radical prostatectomy Gleason score in men with lower PSA levels.

Single-tube multiplex PCR using type-specific E6/E7 primers and capillary electrophoresis genotypes 21 human papillomaviruses in neoplasia

BackgroundHuman papillomavirus (HPV) E6/E7 type-specific oncogenes are required for cervical carcinogenesis. Current PCR protocols for genotyping high-risk HPV in cervical screening are not standardized and usually use consensus primers targeting HPV capsid genes, which are often deleted in neoplasia. PCR fragments are detected using specialized equipment and extra steps, including probe hybridization or primer extension. In published papers, analytical sensitivity is typically compared with a different protocol on the same sample set.A single-tube multiplex PCR containing type-specific primers was developed to target the E6/E7 genes of two low-risk and 19 high-risk genotypes (HPV6, 11 and 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73 and 82) and the resulting short fragments were directly genotyped by high-resolution fluorescence capillary electrophoresis.ResultsThe method was validated using long oligonucleotide templates, plasmid clones and 207 clinical samples of DNA from liquid-based cytology, fresh and formalin-fixed specimens and FTA Microcards® imprinted with cut tumor surfaces, swabbed cervical cancers or ejected aspirates from nodal metastases of head and neck carcinomas. Between one and five long oligonucleotide targets per sample were detected without false calls. Each of the 21 genotypes was detected in the clinical sample set with up to five types simultaneously detected in individual specimens. All 101 significant cervical neoplasias (CIN 2 and above), except one adenocarcinoma, contained E6/E7 genes. The resulting genotype distribution accorded with the national pattern with HPV16 and 18 accounting for 69% of tumors. Rare HPV types 70 and 73 were present as the sole genotype in one carcinoma each. One cervical SCC contained DNA from HPV6 and 11 only. Six of twelve oropharyngeal cancer metastases and three neck metastases of unknown origin bore E6/E7 DNA; all but one were HPV16. One neck aspirate contained atypical squames with HPV26.Analytical sensitivity in dilute plasmid mixes was variable.ConclusionsA primer-rich PCR readily detects the E6/E7 oncogenes of 21 HPV types in cellular and fixed tissue specimens. The method is straightforward, robust and reproducible and avoids post-PCR enzymatic and hybridization steps while detecting HPV with high clinical sensitivity in significant HPV-related neoplasia regardless of specimen type.