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1.  Evaluation of HPV Infection and Smoking Status Impacts on Cell Proliferation in Epithelial Layers of Cervical Neoplasia 
PLoS ONE  2014;9(9):e107088.
Accurate cervical intra-epithelial neoplasia (CIN) lesion grading is needed for effective patient management. We applied computer-assisted scanning and analytic approaches to immuno-stained CIN lesion sections to more accurately delineate disease states and decipher cell proliferation impacts from HPV and smoking within individual epithelial layers. A patient cohort undergoing cervical screening was identified (n = 196) and biopsies of varying disease grades and with intact basement membranes and epithelial layers were obtained (n = 261). Specimens were sectioned, stained (Mib1), and scanned using a high-resolution imaging system. We achieved semi-automated delineation of proliferation status and epithelial cell layers using Otsu segmentation, manual image review, Voronoi tessellation, and immuno-staining. Data were interrogated against known status for HPV infection, smoking, and disease grade. We observed increased cell proliferation and decreased epithelial thickness with increased disease grade (when analyzing the epithelium at full thickness). Analysis within individual cell layers showed a ≥50% increase in cell proliferation for CIN2 vs. CIN1 lesions in higher epithelial layers (with minimal differences seen in basal/parabasal layers). Higher rates of proliferation for HPV-positive vs. -negative cases were seen in epithelial layers beyond the basal/parabasal layers in normal and CIN1 tissues. Comparing smokers vs. non-smokers, we observed increased cell proliferation in parabasal (low and high grade lesions) and basal layers (high grade only). In sum, we report CIN grade-specific differences in cell proliferation within individual epithelial layers. We also show HPV and smoking impacts on cell layer-specific proliferation. Our findings yield insight into CIN progression biology and demonstrate that rigorous, semi-automated imaging of histopathological specimens may be applied to improve disease grading accuracy.
PMCID: PMC4161429  PMID: 25210770
2.  Numerical investigation of two-dimensional light scattering patterns of cervical cell nuclei to map dysplastic changes at different epithelial depths 
Biomedical Optics Express  2014;5(2):485-498.
We use an extensive set of quantitative histopathology data to construct realistic three-dimensional models of normal and dysplastic cervical cell nuclei at different epithelial depths. We then employ the finite-difference time-domain method to numerically simulate the light scattering response of these representative models as a function of the polar and azimuthal scattering angles. The results indicate that intensity and shape metrics computed from two-dimensional scattering patterns can be used to distinguish between different diagnostic categories. Our numerical study also suggests that different epithelial layers and angular ranges need to be considered separately to fully exploit the diagnostic potential of two-dimensional light scattering measurements.
PMCID: PMC3920879  PMID: 24575343
(170.0170) Medical optics and biotechnology; (170.1530) Cell analysis; (170.4580) Optical diagnostics for medicine; (170.4730) Optical pathology; (290.0290) Scattering
3.  Double Staining Cytologic Samples with Quantitative Feulgen-Thionin and Anti–Ki-67 Immunocytochemistry as a Method of Distinguishing Cells with Abnormal DNA Content from Normal Cycling Cells 
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.
Study Design
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.
PMCID: PMC3573880  PMID: 23301387
Ploidy; Early cancer detection; Cervical cancer; Quantitative image cytometry; Proliferation; Immunocytochemistry; Heat-mediated antigen retrieval
4.  Optical Technologies and Molecular Imaging for Cervical Neoplasia: A Program Project Update 
Gender Medicine  2011;9(1 Suppl):S7-S24.
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.
PMCID: PMC3289763  PMID: 21944317
5.  Classifying tissue samples from measurements on cells with within-class tissue sample heterogeneity 
Biostatistics (Oxford, England)  2011;12(4):695-709.
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.
PMCID: PMC3169670  PMID: 21642388
Automating cervical neoplasia screening; Clustering densities; Cumulative log-odds; Functional data clustering; Macro-level classification; Quantitative cytology
6.  Differential expression of miRNAs in the serum of patients with high-risk oral lesions 
Cancer Medicine  2012;1(2):268-274.
Oral cancer is one of the most commonly diagnosed cancers worldwide. Disease is often diagnosed at later stages, which is associated with a poor 5-year survival rate and a high rate of local recurrence. MicroRNAs (miRNAs), a group of small, noncoding RNAs, can be isolated from blood serum samples and have demonstrated utility as biomarkers in multiple cancer types. The aim of this study was to examine the expression profiles of circulating miRNAs in the serum of patients with high-risk oral lesions (HRLs; oral cancer or carcinoma in situ) and to explore their utility as potential oral cancer biomarkers. Global serum miRNA profiles were generated using quantitative PCR method from 1) patients diagnosed with HRLs and undergoing intent-to-cure surgical treatment (N = 30) and 2) a demographically matched, noncancer control group (N = 26). We next honed our list of serum miRNAs associated with disease by reducing the effects of interpatient variability; we compared serum miRNA profiles from samples taken both before and after tumor resections (N = 10). Based on these analyses, fifteen miRNAs were significantly upregulated and five were significantly downregulated based on presence of disease (minimum fold-change >2 in at least 50% of samples, P < 0.05, permutation). Five of these miRNAs (miR-16, let-7b, miR-338-3p, miR-223, and miR-29a) yielded an area under the ROC curve (AUC) >0.8, suggesting utility as noninvasive biomarkers for detection of oral cancer or high-grade lesions. Combining these serum miRNA profiles with other screening techniques could greatly improve the sensitivity in oral cancer detection.
PMCID: PMC3544450  PMID: 23342275
Biomarkers; circulating microRNAs; oral squamous cell carcinoma
7.  Automated sputum cytometry for detection of intraepithelial neoplasias in the lung 
Despite the benefits of early lung cancer detection, no effective strategy for early screening and treatment exists, partly due to a lack of effective surrogate biomarkers. Our novel sputum biomarker, the Combined Score (CS), uses automated image cytometric analysis of ploidy and nuclear morphology to detect subtle intraepithelial changes that often precede lung tumours.
2249 sputum samples from 1795 high-risk patients enrolled in ongoing chemoprevention trials were subjected to automated quantitative image cytometry after Feulgenthionin staining. Samples from normal histopathology patients were compared against samples from carcinoma in situ (CIS) and cancer patients to train the CS.
CS correlates with several lung cancer risk factors, including histopathological grade, age, smoking status, and p53 and Ki67 immunostaining. At 50% specificity, CS detected 78% of all highest-risk subjects—those with CIS or worse plus those with moderate or severe dysplasia and abnormal nuclear morphology.
CS is a powerful yet minimally invasive tool for rapid and inexpensive risk assessment for the presence of precancerous lung lesions, enabling enrichment of chemoprevention trials with highest-risk dysplasias. CS correlates with other biomarkers, so CS may find use as a surrogate biomarker for patient assessment and as an endpoint in chemoprevention clinical trials.
PMCID: PMC3412676  PMID: 22277916
Intraepithelial neoplasia (IEN); lung cancer; risk assessment; intermediate or pre-neoplastic markers and risk factors; biomarkers and intervention studies; chemoprevention; biomarkers and intervention; cancer surveillance and screening; chemoprevention clinical trials; quantitative image cytometry; ploidy analysis; malignancy associated changes
8.  Accuracy of optical spectroscopy for the detection of cervical intraepithelial neoplasia: testing a device as an adjunct to colposcopy 
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.
PMCID: PMC3015005  PMID: 20830707
sensitivity and specificity; diagnosis; early detection of cancer; uterine cervical neoplasms; cervical intraepithelial neoplasia
9.  Epidemiologic Modeling of Cervical Dysplasia with Molecular and Cytopathological Markers 
Gynecologic oncology  2007;107(1 Suppl 1):S163-S169.
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.
PMCID: PMC2978043  PMID: 17727928
human papillomavirus; mRNA; RT-PCR; cytometry; uterine cervix
10.  Potential Use of Quantitative Tissue Phenotype to Predict Malignant Risk for Oral Premalignant Lesions 
Cancer research  2008;68(9):3099-3107.
The importance of early diagnosis in improving mortality and morbidity rates of oral squamous cell carcinoma (SCC) has long been recognized. However, a major challenge for early diagnosis is our limited ability to differentiate oral premalignant lesions (OPLs) at high risk of progressing into invasive SCC from those at low risk. We investigated the potential of Quantitative Tissue Phenotype (QTP), measured by high-resolution image analysis, to recognize severe dysplasia/carcinoma in situ (CIS) (known to have an increased risk of progression) and to predict progression within hyperplasia or mild/moderate dysplasia (termed HMD). We generated a Nuclear Phenotypic Score (NPS), a combination of 5 nuclear morphometric features that best discriminate 4,027 “normal” nuclei (selected from 29 normal oral biopsies) from 4,298 “abnormal” nuclei (selected from 30 SCC biopsies). This NPS was then determined for a set of 69 OPLs. Severe dysplasia/CIS, showed a significant increase in NPS compared to HMD. However, within the latter group, elevated NPS was strongly associated with the presence of high-risk LOH patterns. There was a statistical difference between NPS of HMD that progressed to cancer and those that did not. Individuals with a high NPS had a 10-fold increase in relative risk of progression. In the multivariate Cox model, LOH and NPS together were the strongest predictors for cancer development. These data suggest that QTP could be used to identify lesions that require molecular evaluation and should be integrated with such approaches to facilitate the identification of HMD OPLs at high risk of progression.
PMCID: PMC2693059  PMID: 18451134
Risk prediction; oral malignancy; LOH; dysplasia; image analysis
11.  Up regulation in gene expression of chromatin remodelling factors in cervical intraepithelial neoplasia 
BMC Genomics  2008;9:64.
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.
PMCID: PMC2277413  PMID: 18248679

Results 1-11 (11)