We report here the logistic modeling of the epidemiologic differences between a diagnostic and screening population recruited for the study of optical technologies for cervical cancer detection.
Epidemiologic data were obtained from a risk factor interview as a component of a multicenter Phase II clinical trial which employed fluorescence and reflectance point spectroscopy to diagnose cervical disease. Participants with a recent or past abnormal Papanicolaou smear were grouped into the diagnostic (high-risk) population, while those with a history of normal Papanicolaou smears and no cervical treatments were grouped into the screening (low-risk) population.
Our model revealed that non-white race, greater than a high school education, and peri- and postmenopausal status were associated with the screening population. Meanwhile, a history of genital infections, current OC use, HPV positivity (by Hybrid Capture II and consensus PCR), and histology at clinic visit were important predictors of being in the diagnostic group.
We were successful in recruiting two distinctive populations, and we anticipate being able to use these results to more correctly classify women at higher risk for cervical lesions in our future studies of optical spectroscopy.
cervical dysplasia; epidemiology; optical technologies; risk factors
Both PCR and Hybrid Capture II (HCII) have been used for identifying cervical dysplasia; however, comparisons on the performance between these two tests show inconsistent results. We evaluated the performance of HCII and PCR MY09/11 in both screening and diagnostic populations in sub-sample of 1,675 non-pregnant women from a cohort in three clinical centers in the United States and Canada.
Sensitivity, specificity, positive predictive value, negative predictive value, and concordance between the two tests were calculated.
Specificity of HCII in detecting low-grade squamous intraepithelial lesion (LSIL) was higher in the screening group (88.7%; 95% CI: 86.2%–90.8%) compared to the diagnostic group (46.3%; 95% CI: 42.1%–50.6%); however, specificity of PCR was low in both the screening (32.8%; 95% CI: 29.6%–36.2%) and diagnostic (14.4%; 95% CI: 11.6%–17.6%) groups. There was comparable sensitivity by both tests in both groups to detect high-grade squamous intraepithelial lesion (HSIL); however, HCII was more specific (89.1%; 95% CI: 86.8%–91.0%; 66.2%; 95% CI: 62.0%–70.1%) than PCR (33.3%; 95% CI: 30.2%–36.5%; 17.9%; 95% CI: 14.8%–21.6%) in the screening and diagnostic groups, respectively. Overall agreement for HPV positivity was approximately 50% between HCII and PCR MY09/11; with more positive results coming from the PCR MY09/11.
In the current study, PCR MY09/11 was more sensitive but less specific than HCII in detecting LSIL, and HCII was more sensitive and specific in detecting HSIL than PCR in both screening and diagnostic groups.
comparison; test accuracy; hybrid capture II (HC II); polymerase chain reaction (PCR); cervical dysplasia
Ploidy analysis of Feulgen-thionin stained cervical cytology specimens has been shown to detect cases of high grade cervical dysplasia. However, ploidy analysis alone cannot always distinguish between cells with abnormal DNA content and normal cycling cells. We sought to use double staining with anti-Ki-67 immunocytochemistry to improve ploidy analysis.
Cervical cytology specimens from 49 patients with various diagnoses, mostly dysplasias, from a previous study were used. Samples were double stained with Feulgen-thionin and anti-Ki-67 immunocytochemistry. Ki-67-negative cells were non-cycling, so non-diploid Ki-67-negative cells were likely truly abnormal cells.
The area under the receiver operating characteristic curve for the ability to identify high-grade dysplasias was 0.73 for double staining and 0.74 for thionin-only ploidy analysis on cytospin specimens. At 90% specificity, sensitivities for double staining and thionin alone were 45% and 32%, respectively, but the difference was not statistically significant.
Double staining with Feulgen-thionin and anti-Ki-67 immunocytochemistry does not improve the ability of ploidy analysis of cervical cytology specimens to separate high- and low-grade dysplasias, but our insights into the technical aspects of double staining, especially the effects of antigen retrieval, give hope that this technique could be applied to other immunocytochemical stains that would have a greater ability to improve ploidy analysis.
Ploidy; Early cancer detection; Cervical cancer; Quantitative image cytometry; Proliferation; Immunocytochemistry; Heat-mediated antigen retrieval
There is an urgent global need for effective and affordable approaches to cervical cancer screening and diagnosis. For developing nations, cervical malignancies remain the leading cause of cancer death in women. This reality is difficult to accept given that these deaths are largely preventable; where cervical screening programs are implemented, cervical cancer deaths decrease dramatically. In the developed world, the challenges with respect to cervical disease stem from high costs and over-treatment. We are presently eleven years into a National Cancer Institute-funded Program Project (P01 CA82710) that is evaluating optical technologies for their applicability to the cervical cancer problem. Our mandate is to create new tools for disease detection and diagnosis that are inexpensive, require minimal expertise to use, are more accurate than existing modalities, and will be feasibly implemented in a variety of clinical settings. Herein, we update the status of this work and explain the long-term goals of this project.
Optical Spectroscopy has been studied for biologic plausisbility, technical efficacy, clinical effectiveness, patient satisfaction and cost-effectiveness. We sought to identify healthcare provider attitudes or practices that might act as barriers or to the dissemination of this new technology.
Through an academic-industrial partnership, we conducted a series of focus groups to examine physician barriers to optical diagnosis. The study was conducted in two stages. First, a pilot group of ten physicians (8 obstetrician gynecologists and two family practitioners) was randomly selected from 8 regions of the US and interviewed individually. They were presented with the results of a large trial (N=980) testing the accuracy of a spectroscopy based device in the detection of cervical neoplasia. They were also shown a prototype of the device and were given a period of time to ask questions and receive answers regarding the device. They were also asked to provide feedback of a questionnaire (provided in Appendix A) which was then revised and presented to three larger focus groups (n=13, 15, 17 for a total n=45). The larger focus groups were conducted during national scientific meetings with 20 obstetrician gynecologists and 25 primary care physicians (family practitioners and internists).
When asked about the dissemination potential of the new cervical screening technology, all study groups tended to rely on established clinical guidelines from their respective professional societies with regard to the screening and diagnosis of cervical cancer. In addition, study participants consistently agreed that real-time spectroscopy would be viewed positively by their patients. Participants were positive about the new technology's potential as an adjunct to colposcopy and agreed that the improved accuracy would result in reduced healthcare costs (due to decreased biopsies and decreased visits). However, while all saw the potential of real-time diagnosis, there were many perceived barriers. These barriers included: changes in scheduling and work-flow, liability, documentation, ease of use, length of training, device cost, and reimbursement by third party payers.
Barriers exist to the dissemination of optical technologies into physician practice. These will need to be addressed before cervical screening and diagnosis programs can take advantage of spectroscopy-based instruments for cancer control.
physician attitude; physician satisfaction; dissemination; cervical intraepithelial neoplasia; fluorescence and reflectance spectroscopy; optical spectroscopy
We consider here the problem of classifying a macro-level object based on measurements of embedded (micro-level) observations within each object, for example, classifying a patient based on measurements on a collection of a random number of their cells. Classification problems with this hierarchical, nested structure have not received the same statistical understanding as the general classification problem. Some heuristic approaches have been developed and a few authors have proposed formal statistical models. We focus on the problem where heterogeneity exists between the macro-level objects within a class. We propose a model-based statistical methodology that models the log-odds of the macro-level object belonging to a class using a latent-class variable model to account for this heterogeneity. The latent classes are estimated by clustering the macro-level object density estimates. We apply this method to the detection of patients with cervical neoplasia based on quantitative cytology measurements on cells in a Papanicolaou smear. Quantitative cytology is much cheaper and potentially can take less time than the current standard of care. The results show that the automated quantitative cytology using the proposed method is roughly equivalent to clinical cytopathology and shows significant improvement over a statistical model that does not account for the heterogeneity of the data.
Automating cervical neoplasia screening; Clustering densities; Cumulative log-odds; Functional data clustering; Macro-level classification; Quantitative cytology
Testing emerging technologies involves the evaluation of biologic plausibility, technical efficacy, clinical effectiveness, patient satisfaction, and cost-effectiveness. The objective of this study was to select an effective classification algorithm for optical spectroscopy as an adjunct to colposcopy and obtain preliminary estimates of its accuracy for the detection of CIN 2 or worse. We recruited 1000 patients from screening and prevention clinics and 850 patients from colposcopy clinics at two comprehensive cancer centers and a community hospital. Optical spectroscopy was performed and 4864 biopsies were obtained from the sites measured, including abnormal and normal colposcopic areas. The gold standard was the histologic report of biopsies, read 2–3 times by histopathologists blinded to the cytologic, histopathologic, and spectroscopic results. We calculated sensitivities, specificities, receiver operating characteristic (ROC) curves, and areas under the ROC curves. We identified a cutpoint for an algorithm based on optical spectroscopy that yielded an estimated sensitivity of 1.00 [95% confidence interval (CI) = 0.92 – 1.00] and an estimated specificity of 0.71 [95% CI = 0.62 – 0.79] in a combined screening and diagnostic population. The positive and negative predictive values were 0.58 and 1.00, respectively. The area under the ROC curve was 0.85 (95% CI 0.81 – 0.89). The per-patient and per-site performance were similar in the diagnostic and poorer in the screening settings. Like colposcopy, the device performs best in a diagnostic population. Alternative statistical approaches demonstrate that the analysis is robust and that spectroscopy works as well as or slightly better than colposcopy for the detection of CIN 2 to cancer.
sensitivity and specificity; diagnosis; early detection of cancer; uterine cervical neoplasms; cervical intraepithelial neoplasia
Concerns have been raised that the proportion of cervical cancer preventable by human papillomavirus (HPV) 16/18 vaccines might be lower in sub-Saharan Africa than elsewhere.
In order to study the relative carcinogenicity of HPV types in Nigeria, as well as to estimate the vaccine-preventable proportion of invasive cervical cancer (ICC) in the country, we compared HPV type prevalence among 932 women from the general population of Ibadan, Nigeria, with that among a series of 75 ICC cases diagnosed in the same city. For all samples, a GP5+/6+ PCR based assay was used for the detection of 44 genital HPV types.
In the general population, 245 (26.3%, 95% confidence interval (CI) 23.5% - 29.2%) women were HPV-positive, among whom the prevalence of HPV35 and HPV16 were equally frequent (12.2%, 95% CI 8.4% - 17.0%). In ICC, however, HPV16 predominated strongly (67.6% of 68 HPV-positive cases), with the next most common types being 18 (10.3%, 95% CI 4.2% - 20.1%), 35, 45 and 56 (each 5.9%, 95% CI 1.6% - 14.4%). Comparing among HPV-positive women only, HPV16 and 18 were over-represented in ICC versus the general population (prevalence ratios 5.52, 95% CI 3.7 - 8.3 and 1.4, 95% CI 0.6 - 3.3, respectively). Other high-risk HPV types, as well as low-risk and multiple HPV infections were less common in HPV-positive women with ICC than from the general population.
Our study confirms that in Nigeria, as elsewhere, women infected with HPV16 and 18 are at higher risk of developing ICC than those infected with other high-risk types, and that current HPV16/18 vaccines have enormous potential to reduce cervical cancer in the region.
We compare the racial and ethnic demographics of our participants with the populations where our clinics are located (Texas and British Columbia) and investigate the reasons cited for participation.
We compared the distribution of participants by race/ethnicity to numbers from the 2000 United States Census and the 2001 Census of Canada. Each participant recorded her reasons for enrolling in the trial in her own words. This information was then categorized for analysis. For participants who provided more than one reason for participation, their responses were weighted accordingly to sum 100% for each race. All analyses were performed using SPSS v12.0 (SPSS, Inc, Chicago).
In all, 1,850 women participated in the study. Except for Asians in the Vancouver population and Native Americans in both populations, all minorities were recruited in proportions in excess of their respective proportions in the general population. Distinct differences in the reasons for participating between sites were noted. Houston patients were more likely to cite concern for one's own health as a reason for participating. On the other hand, Vancouver patients were more likely to cite helping others. This trend was found in both the screening and diagnostic populations.
We attribute our success in recruiting minorities to community outreach, our multicultural staff, and efforts to provide uniform care at all sites.
participant recruitment; clinical trials; cervical neoplasia; optical technologies
Conventional cervical screening is insufficient at identifying patients who are likely to progress from cervical dysplasia to carcinoma. Traditional epidemiologic studies have identified potential factors to aid in the discrimination between those lesions likely to progress from those likely to regress; however, there is still much to be learned. To examine the role of traditional epidemiologic factors in conjunction with molecular markers of human papillomavirus activity, we studied a group of women attending colposcopy clinics in Houston, TX and Vancouver, BC between October 2000 and July 2003.
Quantitative real-time PCR was used to measure mRNA expression of the human papillomavirus E7 gene, and quantitative cytology was used to gather information about the DNA index and chromatin features of the cells from these women. Logistic regression was used to establish predictor variables for histologic grade based on the epidemiologic risk factors and the molecular markers.
The most predictive factors were mRNA level, DNA index, parity, and age. The ROC curve for the individual logits indicated excellent discrimination.
In accordance with other authors, these results suggest that molecular markers of the malignant process should be included in analyses looking to predict the progression potential of cervical lesions.
human papillomavirus; mRNA; RT-PCR; cytometry; uterine cervix
We examined intensity and shape differences in 378 repeated spectroscopic measures of the cervix. We examined causes of variability such as presence of precancer or cancer, pathologic tissue type, menopausal status, hormone or oral contraceptive use, and age; as well as technology related variables like generation of device and provider making exam. Age, device generation, and provider were statistically significantly related to intensity differences. Provider and device generation were related to shape differences. We examined the order of measurements and found a decreased intensity in the second measurement due to hemoglobin absorption. 96% of repeat measurements had classification concordance of cervical intraepithelial neoplasia.
(120.0120) Instrumentation, measurement, and metrology; (170.0170) Medical optics and biotechnology; (300.0300) Spectroscopy
In recent decades, extensive resources have been invested to develop cellular, molecular and genomic technologies with clinical applications that span the continuum of cancer care.
In December 2006, the National Cancer Institute sponsored the first workshop to uniquely examine the state of health services research on cancer-related cellular, molecular and genomic technologies and identify challenges and priorities for expanding the evidence base on their effectiveness in routine care.
This article summarizes the workshop outcomes, which included development of a comprehensive research agenda that incorporates health and safety endpoints, utilization patterns, patient and provider preferences, quality of care and access, disparities, economics and decision modeling, trends in cancer outcomes, and health-related quality of life among target populations.
Ultimately, the successful adoption of useful technologies will depend on understanding and influencing the patient, provider, health care system and societal factors that contribute to their uptake and effectiveness in ‘real-world’ settings.
Genomics; Health services research; Emerging technologies; Translational research
To evaluate the performance of the Human Papillomavirus High-Risk DNA test in patients 30 years and older.
Materials and Methods
Screening (N=835) and diagnosis (N=518) groups were defined based on prior Papanicolaou smear results as part of a clinical trial for cervical cancer detection. We compared the Hybrid Capture II® (HCII) test result to the worst histological report. We used cervical intraepithelial neoplasia (CIN) 2/3 or worse as the reference of disease. We calculated sensitivities, specificities, positive and negative likelihood ratios (LR+ and LR−), receiver operating characteristic (ROC) curves, and areas under the ROC curves for the HCII test. We also considered alternative strategies, including Papanicolaou smear, a combination of Papanicolaou smear and the HCII test, a sequence of Papanicolaou smear followed by the HCII test, and a sequence of the HCII test followed by Papanicolaou smear.
For the screening group, the sensitivity was 0.69 and the specificity was 0.93; the area under the ROC curve was 0.81. The LR+ and LR− were 10.24 and 0.34, respectively. For the diagnosis group, the sensitivity was 0.88 and the specificity was 0.78; the area under the ROC curve was 0.83. The LR+ and LR− were 4.06 and 0.14, respectively. Sequential testing showed little or no improvement over the combination testing.
The HCII test in the screening group had a greater LR+ for the detection of CIN 2/3 or worse. HCII testing may be an additional screening tool for cervical cancer in women 30 years and older.
cervical intraepithelial neoplasia; cervix neoplasms; DNA probes HPV; sensitivity and specificity
Development, validation, and implementation of an analytical model to extract biologically and diagnostically relevant parameters from measured cervical tissue reflectance and fluorescence spectra are presented. Monte Carlo simulations of tissue reflectance are used to determine the relative contribution of the signal from the epithelium and stroma. The results indicate that the clinical probe used collects a majority of its reflectance signal from the stroma; therefore, a one-layer analytical model of reflectance is used. Two analytical approaches to calculate reflectance spectra are compared to Monte Carlo simulations, and a diffusion theory-based model is implemented. The model is validated by fitting spectra generated from Monte Carlo simulations and comparing the input and output parameters. Median agreement between extracted optical properties and input parameters is 10.6%. The reflectance model is used together with an analytical model of tissue fluorescence to extract optical properties and fluorophore concentrations from 748 clinical measurements of cervical tissue. A diagnostic algorithm based on these extracted parameters is developed and evaluated using cross-validation. The sensitivity/specificity of this algorithm relative to the gold standard of histopathology per measurement are 85/51%; this is comparable to accuracy reported in other studies of optical technologies for detection of cervical cancer and its precursors.
diffuse reflectance spectroscopy; fluorescence spectroscopy; cancer diagnosis
The highest rates of cervical cancer are found in developing countries. Frontline monitoring has reduced these rates in developed countries and present day screening programs primarily identify precancerous lesions termed cervical intraepithelial neoplasias (CIN). CIN lesions described as mild dysplasia (CIN I) are likely to spontaneously regress while CIN III lesions (severe dysplasia) are likely to progress if untreated. Thoughtful consideration of gene expression changes paralleling the progressive pre invasive neoplastic development will yield insight into the key casual events involved in cervical cancer development.
In this study, we have identified gene expression changes across 16 cervical cases (CIN I, CIN II, CIN III and normal cervical epithelium) using the unbiased long serial analysis of gene expression (L-SAGE) method. The 16 L-SAGE libraries were sequenced to the level of 2,481,387 tags, creating the largest SAGE data collection for cervical tissue worldwide. We have identified 222 genes differentially expressed between normal cervical tissue and CIN III. Many of these genes influence biological functions characteristic of cancer, such as cell death, cell growth/proliferation and cellular movement. Evaluation of these genes through network interactions identified multiple candidates that influence regulation of cellular transcription through chromatin remodelling (SMARCC1, NCOR1, MRFAP1 and MORF4L2). Further, these expression events are focused at the critical junction in disease development of moderate dysplasia (CIN II) indicating a role for chromatin remodelling as part of cervical cancer development.
We have created a valuable publically available resource for the study of gene expression in precancerous cervical lesions. Our results indicate deregulation of the chromatin remodelling complex components and its influencing factors occur in the development of CIN lesions. The increase in SWI/SNF stabilizing molecule SMARCC1 and other novel genes has not been previously illustrated as events in the early stages of dysplasia development and thus not only provides novel candidate markers for screening but a biological function for targeting treatment.
A study was designed to assess variability between different fluorescence spectroscopy devices. Measurements were made with all combinations of three devices, four probes, and thee sets of standards trays. Additionally, we made three measurements on the same day over two days for the same combination of device, probe, and standards tray to assess reproducibility over a day and across days.
Material and Methods
The devices consisted of light sources, fiber optics, and cameras. We measured thirteen standards and present the data from: the frosted cuvette, water, and rhodamine standards. A preliminary analysis was performed with the data that was wavelength calibrated and background subtracted however the system has not been corrected for systematic intensity variations caused by the devices. Two analyses were performed on the rhodamine, water, and frosted cuvette standards data. The first one is based on first clustering the measurements and then looking for association between the 5 factors (device, probe, standards tray, day, measurement number) using chi-squared tests on the cross tabulation of cluster and factor level. This showed that only device and probe were significant. We then did an analysis of variance to assess the percent variance explained by each factor that was significant from the chi-squared analysis.
The data were remarkably similar across the different combinations of factors. The analysis based on the clusters showed that sometimes devices alone, probes alone, but most often combinations of device and probe caused significant differences in measurements. The analysis showed that time of day, location of device, and standards trays do not vary significantly; whereas the devices and probes account for differences in measurement. We expected this type of significance using unprocessed data since the processing corrects for differences in devices. However, this analysis on raw data is useful to explore what combination of device and probe measurements should be targeted for further investigation. This experiment affirms that online quality control is necessary to obtain the best excitation-emission matrices from optical spectroscopy devices.
The fact that the device and probe are the primary sources of variability indicates that proper correction for the transfer function of the individual devices should make the measurements essentially equivalent.
fluorescence spectroscopy devices; quality assurance; trial design; probe; Fast EEM; standards
More than half of the approximately 500,000 women diagnosed with cervical cancer worldwide each year will die from this disease. Investigation of genes expressed in precancer lesions compared to those expressed in normal cervical epithelium will yield insight into the early stages of disease. As such, establishing a baseline from which to compare to, is critical in elucidating the abnormal biology of disease. In this study we examine the normal cervical tissue transcriptome and investigate the similarities and differences in relation to CIN III by Long-SAGE (L-SAGE).
We have sequenced 691,390 tags from four L-SAGE libraries increasing the existing gene expression data on cervical tissue by 20 fold. One-hundred and eighteen unique tags were highly expressed in normal cervical tissue and 107 of them mapped to unique genes, most belong to the ribosomal, calcium-binding and keratinizing gene families. We assessed these genes for aberrant expression in CIN III and five genes showed altered expression. In addition, we have identified twelve unique HPV 16 SAGE tags in the CIN III libraries absent in the normal libraries.
Establishing a baseline of gene expression in normal cervical tissue is key for identifying changes in cancer. We demonstrate the utility of this baseline data by identifying genes with aberrant expression in CIN III when compared to normal tissue.
Few reports of the utilization of an accurate, cost-effective means for measuring HPV oncogene transcripts have been published. Several papers have reported the use of relative quantitation or more expensive Taqman methods. Here, we report a method of absolute quantitative real-time PCR utilizing SYBR-green fluorescence for the measurement of HPV E7 expression in cervical cytobrush specimens.
The construction of a standard curve based on the serial dilution of an E7-containing plasmid was the key for being able to accurately compare measurements between cervical samples. The assay was highly reproducible with an overall coefficient of variation of 10.4%.
The use of highly reproducible and accurate SYBR-based real-time polymerase chain reaction (PCR) assays instead of performing Taqman-type assays allows low-cost, high-throughput analysis of viral mRNA expression. The development of such assays will help in refining the current screening programs for HPV-related carcinomas.
Carcinoma of the cervix is causally related to infection with the human papillomavirus (HPV), and T cells play a pivotal role in the immune response of the host to rid itself of HPV infection. Therefore, we assessed the T-cell function of women with HPV-related cervical neoplasia against a superantigen, Staphylococcus enterotoxin B (SEB). Each woman provided a cervical brush specimen for HPV DNA testing and Papanicolaou (Pap) smears for the staging of cervical lesions. They also provided a blood specimen for determination of the ability of CD4+ T and CD8+ T cells to synthesize Th1 (interleukin-2 [IL-2], gamma interferon [IFN-γ], and tumor necrosis factor alpha [TNF-α]) and Th2 (IL-10) cytokines in response to activation with SEB. Compared with control subjects with self-attested negative Pap smears, women with high-grade squamous intraepithelial lesions (HSIL) had significantly lower percentages of activated CD4+ T cells that produced IL-2 (P = 0.045), IFN-γ (P = 0.040), and TNF-α (P = 0.015) and a significantly lower percentage of activated CD8+ T cells that produced IL-2 (P < 0.01). These data indicate that women with HPV-related cervical HSIL show a decrease in Th1 cytokine production by activated CD4+ T cells and suggested that compromised T-helper functions may negatively impact the function of cytotoxic CD8+ T cells.