Biomedical subjects

Michele Follen

Publications and source records attributed to Michele Follen.

At least 19 recordsLinked to original sources

PubMed2007

Human papillomavirus-related cellular changes measured by cytometric analysis of DNA ploidy and chromatin texture.

BACKGROUND: Image cytometry has provided two highly sensitive markers for the identification of the malignant potential of squamous lesions. Aneuploidy and chromatin texture have been investigated as quantitative measures of nuclear damage in premalignant lesions and carcinoma. Real-time PCR methods have evolved to yield highly specific measurements of mRNA expression in very sparse cellular samples. METHODS: Human papillomavirus (HPV) 16 and 18 E7 mRNA expression was measured using quantitative RT-PCR. DNA index and chromatin measures were taken from image cytology samples. The chromatin features, through discriminant analysis, were aggregated into a score, and both measurements were related to mRNA expression. RESULTS: mRNA level and DNA index show an increasing trend over increasing histological grades. However, DNA index and chromatin score were not correlated to mRNA levels in these samples. Chromatin score differed by mRNA type found with HPV 18 infected samples having a higher score than those with HPV 16. Samples infected with HPV 16 and HPV 18 had even higher chromatin scores. CONCLUSIONS: DNA index and chromatin score were not directly correlated with mRNA levels. However both mRNA and DNA index were related to histological grade, and chromatin score was associated with HPV type. Therefore, DNA index and mRNA levels could be independent predictors of cervical dysplasia, and chromatin score could be related to the viral integration process in cells infected with HPV 18 or dual infections.

Adult↗

PubMed2006

Adenovirus-mediated transfer of human papillomavirus 16 E6/E7 antisense RNA and induction of apoptosis in cervical cancer.

OBJECTIVE: In most cervical cancers, human papillomaviruses (HPVs) are identified. The E6 and E7 genes of HPVs encode proteins, that interfere with the function of the tumor suppressor proteins p53 and Rb. We are exploring the potential use of antisense HPV RNA transcripts for gene therapy for HPV-positive cervical cancers. METHODS: Via a recombinant adenoviral vector, Ad5CMV-HPV 16 AS, we introduced the antisense RNA transcripts of the E6 and E7 genes of HPV type 16 into human cervical cancer SiHa cells harboring HPV 16. We then analyzed the effects of expression of these genes on cell and tumor growth. RESULTS: HPV 16 E6/E7 antisense RNA was detected for 14 days in Ad5CMV-HPV 16 AS-infected cells. After infection, E6 and E7 protein expression was suppressed, and p53 and Rb protein expression increased. The Ad5CMV-HPV 16 AS-infected cells underwent apoptosis in vitro and in vivo. Cell growth and tumorigenicity were greatly suppressed. Ad5CMV-HPV 16 AS treatment significantly reduced the volumes of established subcutaneous tumors. CONCLUSION: Transfection of cervical cancer cells with HPV 16 E6/E7 antisense RNA in a form such as Ad5CMV-HPV 16 AS might be a potentially useful approach to the therapy of HPV 16-positive cervical cancer.

Adenoviridae↗

PubMed2006

Deficiencies in myeloid antigen-presenting cells in women with cervical squamous intraepithelial lesions.

BACKGROUND: There is little information on the function of dendritic cells in women with human papillomavirus (HPV)-related cervical squamous intraepithelial lesions (SILs). In the current study the functions of dendritic cells in the development of T-cell immunity in women with cervical SILs were assessed. METHODS: The percentage of myeloid dendritic cells (MDCs) and plasmacytoid dendritic cells (PDCs) in peripheral blood were enumerated of 44 patients with SIL (low-grade, 19; high-grade, 25), 19 patients with atypical squamous cells of undetermined significance (ASCUS), and 18 controls. The expression of costimulatory receptors was assessed and the ability of monocyte-derived dendritic cells (MDDC) to present HPV16-E6 and HPV16-E7 antigens to autologous T cells. RESULTS: Patients with either low (L)-grade or high (H)-grade SIL had significantly lower median plasma levels of interferon-gamma than did the controls (P = .038 and .031, respectively). Compared with the controls, patients with ASCUS or LSILs had significantly lower median percentages of MDCs (P = .002 and P < .001, respectively), and significantly lower median percentages of MDDCs that expressed CD86 (P < .001 and P = .003, respectively) and major histocompatability complex class-II antigen human leukocyte antigen DR (HLA-DR) (P = .012 and P < .001, respectively). T cells of patients with ASCUS or LSILs proliferated less than those of the controls in response to HPV16-E7 (P = .002 and .046, respectively). CONCLUSIONS: Low levels of peripheral blood MDCs and of MDDCs expressing CD86 and HLA-DR suggest that deficiencies in the ability of MDDC to present antigen to autologous T cells may lead to persistent infection with HPV and the development of cervical SILs in HPV-infected women.

Adult↗

PubMed2006

DNA ploidy compared with human papilloma virus testing (Hybrid Capture II) and conventional cervical cytology as a primary screening test for cervical high-grade lesions and cancer in 1555 patients with biopsy confirmation.

BACKGROUND: Because 80% of cervical cancers arise in low-resource settings, many inexpensive strategies are being tested. In that spirit, the authors are testing large-scale genomic or DNA ploidy measurements as an inexpensive and semiautomated strategy. METHODS: Patients entered either a screening or diagnostic study of several optical technologies: quantitative cytology, quantitative histopathology, and fluorescence and reflectance spectroscopy using a point probe, a multispectral digital colposcope, or a combination of the two. We calculated sensitivities, specificities, positive and negative predictive values, and their confidence interval testing conventional cytology, Hybrid Capture (HC) II testing, and DNA ploidy measured on the Feulgen-stained quantitative Pap smear. RESULTS: The current investigation reports on 1555 patients for whom colposcopically directed biopsies were read 3 times by study pathologists. The final histopathologic diagnosis was high grade (cervical intraepithelial neoplasia [CIN] 2, CIN 3, carcinoma in situ [CIS], and cancer) in 16% of patients. Using high-grade squamous intraepithelial lesions (SILs) histopathology as the threshold and gold standard, the sensitivity and specificity, respectively, were: 0.47 and 0.96 for conventional cytology, 0.91 and 0.80 for HC II, and 0.59 and 0.93 for DNA ploidy. The positive and negative predictive values (PPV, NPV) for conventional cytology were 0.70 and 0.90, 0.46 and 0.98 for HC II, and 0.63 and 0.92 for DNA ploidy. CONCLUSIONS: DNA ploidy shows comparable sensitivity, specificity, PPV, and NPV values to conventional cytology and HC II. Unlike conventional cytology, DNA ploidy is semiautomated and can be performed in less than 8 hours. Cost effectiveness studies are under way, but in the authors' laboratory DNA ploidy is inexpensive.

Adolescent↗

PubMed2006

Progress in chemoprevention drug development: the promise of molecular biomarkers for prevention of intraepithelial neoplasia and cancer--a plan to move forward.

This article reviews progress in chemopreventive drug development, especially data and concepts that are new since the 2002 AACR report on treatment and prevention of intraepithelial neoplasia. Molecular biomarker expressions involved in mechanisms of carcinogenesis and genetic progression models of intraepithelial neoplasia are discussed and analyzed for how they can inform mechanism-based, molecularly targeted drug development as well as risk stratification, cohort selection, and end-point selection for clinical trials. We outline the concept of augmenting the risk, mechanistic, and disease data from histopathologic intraepithelial neoplasia assessments with molecular biomarker data. Updates of work in 10 clinical target organ sites include new data on molecular progression, significant completed trials, new agents of interest, and promising directions for future clinical studies. This overview concludes with strategies for accelerating chemopreventive drug development, such as integrating the best science into chemopreventive strategies and regulatory policy, providing incentives for industry to accelerate preventive drugs, fostering multisector cooperation in sharing clinical samples and data, and creating public-private partnerships to foster new regulatory policies and public education.

Biomarkers, Tumor↗

PubMed2005

Correlates of cervical mucosal antibodies to human papillomavirus 16: results from a case control study.

BACKGROUND: While the cervical mucosal immune response to human papillomavirus (HPV) infection is believed to be central to viral clearance, it is not well characterized. We performed this analysis to determine correlates of HPV-16-specific mucosal antibody response in women at high risk for infection with HPV. METHODS: Cervical mucosal and serum samples were obtained from participants in a case control study that measured demographic risk factors of cervical disease and HPV infection. An HPV-16 L1-virus-like particle ELISA was used to detect HPV-16-specific IgA and IgG. Antibody level results were correlated with demographic characteristics, sexual history, cervical disease, and HPV detection. RESULTS: Cervical anti-HPV-16 IgA and IgG inversely correlated with HPV DNA, HPV-16 DNA, and cervical disease. CONCLUSIONS: These findings suggest that mucosal antibodies may protect against HPV infection and cervical disease. However, additional longitudinal studies evaluating serum and mucosal antibody correlates of incident, persistent, and clearing HPV infection are needed. In addition, standardization of mucosal sample collection and testing methods are required.

Adolescent↗

PubMed2005

Interactive dynamic graphical techniques for the exploration of functional data.

INTRODUCTION: The use of interactive dynamic graphics has become a common practice for the exploration of multidimensional data sets. The availability of powerful and inexpensive hardware and software for graphical computing makes the use of such techniques feasible for the examination of complex forms of data. This paper describes some simple techniques, which were implemented in the LISP-STAT environment, for the visualization of functional data arising from studies of optical technologies used for the detection of cervical intraepithelial neoplasia or squamous intraepithelial lesions. MATERIALS AND METHODS: The methods demonstrated have been implemented in software coded in LISP-STAT, a free statistical computing package available for most computer systems. The data used in this paper are drawn from a previous study in which fluorescence spectroscopy was measured from cervical sites at 337 nm, 380 nm, and 460 nm excitation in cervical screening patients. The goal of the project is to explore biographical variables to better understand the biology of fluorescence. RESULTS: 199 measurements were taken in 55 women with normal Pap smears. The data are recorded as spectra showing the intensity of emission excitation versus emission in nanometers. Covariate variables available for analysis are current smoker vs. nonsmoker premenopausal vs. postmenopausal, tissue type (columnar, squamous, and transition zone), and age in years. Although the optical measurements show consistent changes between normal and abnormal tissue in individual patients, there is wide variation in the intensity of the measurements between patients, even for normal tissue. Patient age affects the fluorescent spectrum showing increasing intensity with increasing age. Menopausal status affects the fluorescent spectra coincidentally with age. Smoking and race do not appear to affect the spectra in this sample of patients. CONCLUSIONS: The use of interactive graphical techniques permits the data analyst to examine multidimensional data in intuitive ways. These explorations allow non-statisticians to explore the data in a perceptive manner that may lead to new approaches in algorithm development for optical technologies.

Analysis of Variance↗

PubMed2005

Immune responses to repetitive adenovirus-mediated gene transfer and restoration of gene expression by cyclophosphamide or etoposide.

BACKGROUND: One major concern about adenoviral vectors for repetitive gene delivery is the induction of an immune response to the vector, thus impeding effective gene transduction. METHODS: To assess the immune response to the adenoviral vector, repetitive gene dosing was performed into rhesus monkey cervix and C3H mouse skin using the adenoviral vector carrying the lacZ gene. Three repetitive intracervical injections of adenovirus-lacZ were done in the rhesus monkey at the intervals of 4 weeks. Gene expression on the second and third injection was completely suppressed. RESULTS: Anti-adenovirus IgG levels and neutralizing antibody titers in the rhesus monkey significantly increased after the first injection of adenovirus. In the C3H mouse, neutralizing antibody titers significantly increased after the first injection of adenovirus-lacZ at more than 10(8) plaque-forming unit (PFU). The repetitive expression of lacZ gene in the mouse skin markedly decreased when the second injection is done more than 2 weeks after the first injection. Chronic low-dose treatment with cyclophosphamide or etoposide markedly suppressed neutralizing antibody titers in the mouse serum and restored the gene expression in the mouse skin on the second and third injection. CONCLUSIONS: It is suggested that repetitive gene expression by adenovirus-mediated transfer may be reduced by circulating neutralizing antibodies and could be restored by chronic low-dose treatment with cyclophosphamide or etoposide.

Adenoviridae↗

PubMed2005

Results of a pilot study of multispectral digital colposcopy for the in vivo detection of cervical intraepithelial neoplasia.

OBJECTIVE: Fluorescence spectroscopy is a promising technology for the detection of cervical squamous intraepithelial precancers and cancers. To date, many investigators have focused on point spectroscopy as an adjunct to diagnostic colposcopy. A device that visualizes the whole field of the cervix is needed for screening. To that end, we have developed a multispectral digital colposcope that works through the colposcope to image with white light, UV excitation at 345 nm, and blue light at 440 nm excitation. Here, we report the pilot study that precedes a Phase I trial. METHODS: The MDC system is composed of a light source, a colposcope, and a video rate color CCD camera with a frame grabber and takes approximately less than 1 min to make images of the cervix. Patients were measured at baseline and after acetic acid placement with white light, 345 nm excitation, and 440 nm excitation from the xenon arc lamp. The white light is in the visible spectrum, 345 nm excitation is in the UV spectrum and is not visible, and 440 nm excitation is blue light in the visible spectrum. White light generates a pink image of the cervix. 345 nm excitation, the UV light, excites fluorophores to emit a blue image. 440 nm excitation, the blue light, excites fluorophores to emit a green image. The patients underwent a loop excision procedure and the histopathology was inked and cut into 12 sections by the study pathologists. The histopathologic slides were scanned and the images were then reconstructed into maps. A diagnostic algorithm was calculated. The data were preprocessed, transformed, and analyzed by the K-means clustering method. Disease maps were generated using the algorithm and classifier and compared to white light colposcopy and the blue and green images obtained at 345 and 440 nm. RESULTS: Forty-six patients were measured at four clinical sites. Images were made of the cervix with white light, 345 nm excitation, and 440 nm excitation and are presented in the figures. As the study went on, images improved with improvements in the instrument. The white light and fluorescence images are presented with crudely constructed histopathologic maps and algorithmic maps. At 345 nm excitation, the UV light, histologically confirmed CIN appears darker blue; while at 440 nm excitation, the blue light, histologically confirmed CIN appears lighter green. CONCLUSIONS: This pilot study shows that MDC images can be matched to both histopathologic and algorithmic maps. The device and the algorithm are evolving but show promise. A Phase I trial is planned.

Adult↗

PubMed2005

Evolving a strategic approach to cervical cancer control in Africa.

Nigeria is a country in Western Africa where the incidence rate of cervical cancer is 25/100,000. There are 32 million women aged 15-64 years old. If we were to conduct a one-time screen over 1 year, 8000 new invasive cervical cancers would be detected. Currently, 80% of cases present in Stage III. While a one-time screen should detect some earlier stages, there could be as many as 6400 Stage III cancers to treat. Strategies to enable a one-time screen are being considered.

Adult↗

PubMed2005

Subvisual chromatin changes in cervical epithelium measured by texture image analysis and correlated with HPV.

OBJECTIVES: In this study, we are testing the hypothesis that human papillomavirus (HPV) positivity is correlated with chromatin texture in the cell. Interim analyses are important since this study involves 2000 patients and generates 6000 biopsy specimens that will be subjected to quantitative histopathological analysis and correlated to HPV positivity as measured by the Hybrid Capture II test (Digene; Gaithersberg, MD) and both HPV-DNA and mRNA by the polymerase chain reaction (PCR). The studies of optical technologies, from which we derive this sample, use the colposcopically directed and histopathologically classified cervical biopsy as the gold standard. In this report, we describe the results of an interim analysis of quantitative histopathology and chromatin texture as correlates of HPV infection using the cyto-savant system in cytologically and histopathologically negative specimens. METHODS: A group of 1544 patients entered the optical technology trials, generating 3275 biopsies and 1544 Papanicolaou readings. Two hundred forty-eight patients were cytologically and histopathologically negative. Study pathologists reviewed histologic samples 3 times in a blinded fashion. Non-overlapping, quantitatively stained nuclei were selected from the samples by the pathologists. HPV testing was done using the PCR method and the Hybrid Capture II test. Statistical analysis involved the creation of a classification matrix using a linear discriminant analysis. The matrix was trained on HPV-positive cells by PCR. The analysis included the random creation of both a training set and a validation set that were classified based on the discrimination score obtained by correlating nuclear texture with HPV positivity. RESULTS: The sensitivity of the classification was 52-54% and the specificity was 77-78%. Overall, a 68% predicted accuracy was achieved for both the training set and the test set. The agreement of a test and training set shows that the sets created randomly are indeed similar, and that the discrimination score worked equally well in both sets of cells. Once a cell-by-cell algorithm for HPV positivity was derived, HPV positivity was recalculated on the basis of cell-by-cell texture features. HPV positivity was then recalculated on both a per-biopsy basis and a per-patient basis. For HPV 16 and 18, the positivity rate was 70% on a per-biopsy basis and 73% on a per-patient basis. CONCLUSIONS: Although these results are preliminary, they suggest that texture features reflecting chromatin condensation may correlate with HPV positivity. The current sample is histologic, the analysis suggests that in a cytologic sample, HPV positivity could be detected or confirmed by texture features computed as part of an HPV-associated score. Additional biologic markers could be used as needed. While this study was performed on histologic samples, a study of cytologic samples would be more useful. Future studies will examine chromatin texture compared to HPV integration and mRNA HPV expression.

Adolescent↗

PubMed2005

Classification using the cumulative log-odds in the quantitative pathologic diagnosis of adenocarcinoma of the cervix.

INTRODUCTION: This study develops a method that discriminates between normal and cancerous tissue sections (i.e., populations of cells) using a statistical model applied to high-dimensional quantitative measurements made on a sample of cells. MATERIALS AND METHODS: We use a cumulative log-odds model to create a score for a tissue section using the information from the cells within that tissue section. Then, a threshold is determined using receiver operating characteristic (ROC) curve analysis. The method was tested using data from cervical adenocarcinomas, adenocarcinoma in situ, and normal columnar tissue. RESULTS: Using 120 potential features, we analyzed the data for staining-independent features. Twenty-two features were statistically significant. We then calculated the log-odds and created a score, followed by ROC curve analysis. The operating point which maximizes the sum of the specificity and sensitivity achieved a sensitivity of 100% with a specificity of 85%. CONCLUSION: The cumulative log-odds performs well in classifying tissue sections using high-dimensional data measured at the cellular level, like that of quantitative pathology. This methodology potentially has applications in pathology, radiology, and optical technologies.

Adenocarcinoma↗

PubMed2005

Design and preliminary analysis of a study to assess intra-device and inter-device variability of fluorescence spectroscopy instruments for detecting cervical neoplasia.

INTRODUCTION: A study was designed to assess variability between different fluorescence spectroscopy devices. Measurements were made with all combinations of three devices, four probes, and three sets of standards trays. Additionally, we made three measurements on the same day over 2 days for the same combination of device, probe, and standards tray to assess reproducibility over a day and across days. MATERIALS 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 were 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. RESULTS: 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. CONCLUSION: 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.

Analysis of Variance↗

PubMed2005

The nude rat as an orthotopic model for cervical cancer.

OBJECTIVE: The purposes of this study were to establish intracervical tumors of the nude rat as an orthotopic experimental model for human cervical cancer and to preliminary evaluate the effects of the adenoviral vector, Ad5CMV-p53, on orthotopic cervical tumor size. METHODS: Human cervical cancer SiHa and ME-180 cells were injected into the cervix of the nude rat. Four days later, 1 x 10(9) plaque forming units (PFU) of Ad5CMV-p53 were injected into the cervix. The rats were later sacrificed to determine cervical tumor size. RESULTS: Eight of ten nude rats developed SiHa cell tumors; all ten nude rats developed ME-180 cell tumors. Four of ten SiHa cell tumors metastasized to the pelvic cavity; no ME-180 cell tumors did. The growth of Ad5CMV-p53-infected cells was greatly suppressed. The ad5CMV-p53 treatment significantly reduced both cell tumor volumes in nude rat cervixes. CONCLUSION: The nude rat cervix grows tumors similar to human cervical cancer tumors and makes an excellent experimental model. Transfection of cervical cancer cells with the wild-type p53 gene via Ad5CMV-p53 is a potential therapeutic approach to cervical cancer.

Adenoviridae↗

PubMed2005

Inverse correlation of cellular immune responses specific to synthetic peptides from the E6 and E7 oncoproteins of HPV-16 with recurrence of cervical intraepithelial neoplasia in a cross-sectional study.

BACKGROUND: Epidemiological studies have clearly established that human papillomavirus (HPV) infection is the major risk factor for cervical cancer. Most cervical cancers and pre-cancers are HPV-positive. Not all pre-cancers progress to cancer; a significant number regress. The immunological basis for either spontaneous or treatment-mediated recovery from HPV-associated CIN is not clear. Currently, prophylactic vaccines are successfully inducing antibody responses in HPV negative patients. Therapeutic vaccines for HPV-positive patients with disease are needed. There is a need to understand the immunologic basis for the Cell-Mediated Immune (CMI) response and for histological regression to help the formulation of therapeutic vaccines. MATERIAL AND METHODS: Four groups of women were identified for this cross-sectional study of CMI. Group 1 consisted of six women without cytological or histological diagnosis of CIN and with an HPV negative test (CIN((-))/HPV((-))). Group 2 included 31 women with a new histological diagnosis of CIN and HPV positive test (CIN((+))/HPV((+))). Groups 3 and 4 were selected from women who had undergone ablative or excisional treatment for CIN at the colposcopy clinic at least 6 months before the study. The women in groups 3 and 4 were (CIN((+))/HPV((+))) before CIN treatment. Group 3 consisted of 22 women without evidence of recurrence of CIN (Recur((-))), and group 4 included 10 with histological diagnosis of recurrent CIN (Recur((+))). In particular, we investigated CMI responses to synthetic peptides from the E6 and E7 oncoproteins of HPV-16. RESULTS: Compared to patients with disease recurrence (Recur((+)), n = 10), the majority of individuals who remained recurrence-free post-treatment (Recur((-)), n = 22) exhibited significant proliferative responses to synthetic peptides from the E6 (P = 0.001) and the E7 (P = <0.001). In particular, significant responses were observed with the E6 peptide Q15L (aa 43-57, P = 0.006) and the E7 peptide Q19D (aa 44-62, P = 0.002) in Recur((-)) patients but not Recur((+)) individuals. Additionally, PBMC from women in the Recur((-)) group, but not the Recur((+)) group, produced predominantly TH1 cytokines upon stimulation with the peptides Q15L or Q19D. CONCLUSIONS: These results indicate an association between significant cellular immune responses specific to synthetic peptides from the E6 and E7 oncoproteins of HPV-16 and recurrence-free survival in HPV patients treated for CIN. We predict that these peptides may be useful as indicators of protective immunity for recovery from CIN and also for potential inclusion in designing immunotherapeutic and immunoprophylactic reagents for HPV-associated CIN.

Adolescent↗

PubMed2005

Kappa statistics to measure interrater and intrarater agreement for 1790 cervical biopsy specimens among twelve pathologists: qualitative histopathologic analysis and methodologic issues.

BACKGROUND: As part of a program project to evaluate emerging optical technologies for cervical neoplasia, we performed fluorescence and reflectance spectroscopic examinations of patients with abnormal Papanicolaou smears. Biopsy specimens were taken from each area and measured optically, and study pathologists performed qualitative histopathologic readings. Several methodologic issues arose in this analysis: (1) the interpathologist and intrapathologist agreement between institutions for the 1790 biopsy specimens; (2) the interinstitutional agreement among the two institutions conducting the trials on 117 randomly chosen biopsy specimens; (3) the interinstitutional agreement among the two institutions and a third expert gynecologic pathologist to ensure the expert readings were comparable to those outside both institutions on 117 randomly chosen biopsy specimens; and (4) an additional three reviews of the 106 difficult biopsy specimens by all three institutions. METHODS: All 1790 specimens from 850 patients were reviewed three times at each institution in blinded fashion; those for which the first and second reviews were identical were not reviewed a third time. A randomly selected sample of 117 specimens was randomly ordered and read by study pathologists at The University of Texas M. D. Anderson Cancer Center, British Columbia Cancer Agency (BCCA), and Brigham and Women's Hospital (BWH). The 106 difficult cases were treated in the same manner as the randomized and random-ordered cases. Generalized, unweighted, and weighted kappas and their 95% confidence intervals were used to assess agreement. Binary comparisons were used to compare diagnostic categories. FINDINGS: The kappas for the three readings of the overall data set using eight-category World Health Organization (WHO) criteria were as follows: 0.66 for the generalized, 0.72 for weighted, and ranged from 0.59 to 0.94 unweighted binary categories; those read using four-category Bethesda criteria: 0.70 for generalized, 0.69 for weighted, and 0.56-0.94 for unweighted binary categories. For the pool versus the study pathologist readings, the eight-category kappa was 0.51 for generalized, 0.72 for weighted, and 0.56-0.82 for unweighted binary categories; for those read using Bethesda criteria: 0.70 for generalized, 0.70 for weighted, and 0.59-0.82 for the unweighted binary categories. The interpathologist and intrapathologist readings were fair by Landis standards at the low end of the diagnostic scale (atypia, human papillomavirus, and CIN1) and substantial to almost perfect at the high end (CIN2, CIN3, and CIS). The randomly selected and randomly ordered sample of 117 specimens read with the WHO system yielded a generalized kappa of 0.45; among the three institutions (M. D. Anderson Cancer Center vs. BCCA, M. D. Anderson vs. BWH, and BCCA vs. BWH), the unweighted kappas were 0.46, 0.41, and 0.49 and the weighted were 0.65, 0.66, and 0.68, respectively; for the Bethesda, a generalized kappa of 0.65, unweighted kappas of 0.66, 0.65, and 0.47, and weighted of 0.74, 0.72, and 0.74. The difficult specimens read with the WHO system yielded a generalized kappa of 0.23; among the three institutions the unweighted kappas were 0.20, 0.30, and 0.37, and the weighted were 0.17, 0.34, and 0.31; for the Bethesda, a generalized kappa of 0.25; among the three institutions, the unweighted kappas were 0.21, 0.32, and 0.37, and the weighted were: 0.07, 0.21, and 0.37, respectively. INTERPRETATION: Kappas in this expert group of pathologists were in the moderate, substantial, and almost perfect ranges for the overall and randomized samples. The randomized sample was representative of the larger sample. The kappa of the specimens for which disagreements arose was, predictably, in the slight range. Our findings will aid both the correlations with optical measurements using fluorescence and reflectance spectroscopy and the quantitative histopathologic analysis of these study specimens.

Biopsy↗

PubMed2005

Diffuse reflectance patterns in cervical spectroscopy.

OBJECTIVES: Our laboratory seeks to develop minimally invasive cost-effective methods to improve screening and detection of curable precursors to cervical cancer. Previously, we have presented pilot studies that assess the diagnostic power of auto-fluorescence and diffuse reflectance spectroscopy. In the present study, we evaluate diffuse reflectance spectra from a comprehensive 850 patient clinical trial to determine its ability to discriminate normal tissue from several grades of abnormal cervical tissue. METHODS: Diffuse reflectance spectra at four source detector separations measured from 549 cervical sites were available for analysis. Three classifiers were implemented: one used spectral data directly as input, a second used simple spectral features such as peak position and intensity, and one used principal component analysis for feature selection. Algorithms were developed and evaluated using leave-one-out cross-validation to classify normal and precancerous cervical tissue. The percentage of samples correctly classified was used to evaluate and compare the performance of the algorithms, as compared to histology. RESULTS: Diffuse reflectance spectra of cervical precancer showed consistent differences from that of normal tissue at all source detector separations; reflectance intensity of precancer was lower than that of normal tissue on average. Normal cervical tissue spectra show more intensity variation between patients than other tissue grades. Reflectance spectra acquired from the closest source detector separations consistently demonstrated the most relevant information for tissue classification. Two persistent spectral patterns demonstrated that the contribution of hemoglobin absorption and the wavelength-dependent spectral slope contained relevant information for classification. CONCLUSIONS: Spectral patterns in diffuse reflectance spectra can be used for the discrimination of normal cervical tissue from low grade and high grade squamous intraepithelial lesions.

Algorithms↗

PubMed2005

Cervical chromosome 9 polysomy: validation and use as a surrogate endpoint biomarker in a 4-HPR chemoprevention trial.

BACKGROUND: Several genetic alterations have been described in cervical cancers including: human papillomavirus (HPV) E6 and E7 oncoproteins, subtle sequence changes, alterations in chromosome number, chromosome translocations, and gene amplifications. This report focuses on establishing chromosome 9 polysomy as a cervical biomarker of chromosome instability and using it in a chemoprevention trial. Chromosomal instability is a feature of most human cancers and is probably an early event in the process. METHODS: We used 37 cervical cone specimens to validate chromosome 9 polysomy as a biomarker and then tested its modulation in a randomized clinical trial of 4-hydroxyphenylretinamide (4-HPR) in 39 patients with three blinded histopathologic reviews. No confounders were identified. In the present study, immunohistocytochemical analysis of Chromosome 9 polysomy was carried out and quantitatively measured. RESULTS: The Cell Index, the ratio of the number of total chromosome 9 copies to the total number of ells, increases significantly in archival samples as the cervix changes from normal to CIN to invasive cancer. In the chemoprevention trial, chromosome 9 polysomy was used as a biomarker and supported the histological analysis showing that 4-HPR impaired the natural regression response. CONCLUSIONS: Chromosome 9 polysomy appears to be a marker of genetic instability that can be used in chemoprevention trials as a surrogate endpoint biomarker. In this randomized trial of 4-HPR, the chromosome 9 polysomy measurements supported the clinical histopathologic reading in a quantitative manner suggesting that 4-HPR at 200 mg/day may have been inhibiting the regression seen in the placebo arm by inducing genetic instability.

Aneuploidy↗