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.

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.

Optical coherence tomography: a pilot study of a new imaging technique for noninvasive examination of cervical tissue.

OBJECTIVE: Optical coherence tomography (OCT) is a novel noninvasive technique that can map subsurface tissue structure with a resolution of 10 to 20 mum. The objective of this study was to determine whether an OCT imaging system could be used clinically in vivo to image and distinguish features of normal and abnormal cervical tissue. STUDY DESIGN: Cervical OCT images and biopsy specimens were obtained from consenting volunteers. Images were analyzed quantitatively for intensity of backscattered light from the epithelia and for rates of signal decay of signal over the depth of epithelia (slope). Patients were stratified by menopausal status, and parameters were compared in normal and abnormal cervical samples, as diagnosed by routine histopathologic techniques. RESULTS: Average epithelial intensities were significantly stronger in the abnormal tissue than in the normal tissue of premenopausal women (P<.0024), but were stronger in the normal tissue of postmenopausal women (P<.062). No significant differences in signal decay rate were detected. CONCLUSION: OCT images, which contain information about epithelial and stromal structure, can be clinically obtained. Image features of normal and abnormal cervical epithelium differ significantly.

Results of a phase II double-blinded randomized clinical trial of difluoromethylornithine for cervical intraepithelial neoplasia grades 2 to 3.

PURPOSE: Our purpose was to conduct a double-blinded randomized trial of difluoromethylornithine (DFMO) at 0.125, 0.5 gm/m2, versus placebo in the treatment of cervical intraepithelial neoplasia (CIN) grades 2 to 3. A promising phase I study has shown histopathologic responses at these dose levels. EXPERIMENTAL DESIGN: Patients with histopathologically confirmed CIN 2-3 lesions were recruited from a colposcopy clinic and underwent Papanicolaou testing, human papillomavirus testing, and colpophotography. They took oral contraception and DFMO or placebo elixir for 28 days and filled out the National Cancer Institute common toxicity calendars. They returned for follow-up and a repeat Papanicolaou smear, colpophotograph, and loop excision of the cervix. RESULTS: There were no statistically significant differences among the arms in histopathologic response. This could no be explained by any biases in risk factors. The prominent toxicities were diarrhea, dizziness, nausea, and headaches. There were no differences in the toxicities among arms. The Papanicolaou smear was a poor biomarker of response and correlated poorly with the histopathology. CONCLUSIONS: DFMO is no active at 0.125 and 0.5 gm/m2 for 28 days when given orally in CIN 2-3. Higher oral doses or longer administration is necessary, supporting data from breast trials. Alternatively, a trial of topical DFMO might merit attention as activity has been noted in trials of actinic keratoses.

Quantitative histopathology and chromosome 9 polysomy in a clinical trial of 4-HPR.

OBJECTIVE: This trial examined the use of 4-hydroxyphenyl-retinamide (4-HPR), demonstrated to be a potent inhibitor of carcinogenesis in vitro and in animal models, in patients with cervical intraepithelial neoplasia (CIN) grades 2 to 3. Quantitative pathology and chromosome 9 polysomy were used to understand the biology and quantify the clinical histopathologic changes observed. METHODS: Patients were randomized to 4-HPR or placebo for 6 months and followed for six more months. Cervical biopsies were obtained at baseline, 6 months, and 12 months; the biopsies were read blinded three times by the study pathologist. Feulgen-stained sections were also obtained and analyzed using computer-assisted image cytometry. Chromosome 9 polysomy was performed on tissue slices using in situ hybridization and measured quantitatively. Statistical analyses were carried out in S-Plus (Insightful Corporation, Seattle, WA) and R. RESULTS: The interim analysis, planned for 40 patients, was carried out on 39. The 6- and 12-month analyses showed a statistically significant difference between the two study arms. When code was broken, the 4-HPR-treatment arm was found to have fared less well than placebo. Analyses of Feulgen-stained sections provided a quantitative measure of the increase of DNA content and texture features. Chromosome 9 polysomy was also measured using image analysis. The changes observed were consistent with those of cells displaying cancerous changes, indicating a lack of response. CONCLUSION: 4-HPR is not active at 200 mg/day. The interim analysis was helpful in directing the study; and, in this case, ending it. The intermediate endpoint biomarkers of quantitative histomorphometry and chromosome 9 polysomy yielded quantitative and repeatable results consistent with the findings of the clinical pathologist.

Use of nuclear morphometry characteristics to distinguish between normal and abnormal cervical glandular histologies.

This is a methodological study exploring the use of quantitative histopathology applied to the cervix to discriminate between normal and cancerous (consisting of adenocarcinoma and adenocarcinoma in situ) tissue samples. The goal is classifying tissue samples, which are populations of cells, from measurements on the cells. Our method uses one particular feature, the IODs-Index, to create a tissue level feature. The specific goal of this study is to find a threshold for the IODs-Index that is used to create the tissue level feature. The main statistical tool is Receiver Operating Characteristic (ROC) curve analysis. When applied to the data, our method achieved promising results with good estimated sensitivity and specificity for our data set. The optimal threshold for the IODs-Index was found to be 2.12.

Light scattering from cervical cells throughout neoplastic progression: influence of nuclear morphology, DNA content, and chromatin texture.

A number of noninvasive fiber optic optical technologies are under development for real-time diagnosis of neoplasia. We investigate how the light scattering properties of cervical cells are affected by changes in nuclear morphology, DNA content, and chromatin texture, which occur during neoplastic progression. We used a Cyto-Savant computer-assisted image analysis system to acquire quantitative nuclear features measurements from 122 Feulgen-thionin-stained histopathologic sections of cervical tissue. A subset of the measured nuclear features was incorporated into a finite-difference time-domain (FDTD) model of cellular light scattering. The magnitude and angular distribution of scattered light was calculated for cervical cells as a function of pathologic grade. The nuclear atypia strongly affected light scattering properties. The increased size and elevated DNA content of nuclei in high-grade lesions caused the most significant changes in scattering intensity. The spatial dimensions of chromatin texture features and the amplitude of refractive index fluctuations within the nucleus impacted both the angular distribution of scattering angles and the total amount of scattered light. Cellular scattering is sensitive to changes in nuclear morphology that accompany neoplastic progression. Understanding the quantitative relationships between nuclear features and scattering properties will aid in the development of noninvasive optical technologies for detection of precancerous conditions.

Cytometric features of cell nuclei of adenocarcinoma in situ and invasive adenocarcinoma of the cervix.

OBJECTIVE: The goal of this study was to characterize adenocarcinoma in situ (ACIS) and invasive adenocarcinoma (AdCa) of the cervix by using image histometric measurements of nuclear morphometric features. STUDY DESIGN: Archival pathology slides and tissue blocks from 37 patients with ACIS, 18 with invasive AdCa, and 13 with normal cervical epithelial and glandular histology were reviewed by two pathologists. The controls were matched for age and menstrual status and as closely as possible for the age of the slides; this limited the number of normal cases available. Morphometric, photometric, and textural measurements were made on 4-microm sections of tissue stained with a thionin-SO(2) Feulgen reaction. A mixed analysis of covariance model was used for analysis. RESULTS: The Integrated Optical Density Index was found between the mean value for normal cells and that for ACIS and invasive AdCa (P <.001). Twenty-two other morphometric features were identified that exhibited differences in their means between at least two of the three tissue types. CONCLUSION: In the cell populations studied, certain nuclear image features were found to correlate with histologic diagnosis. The features can be measured objectively and could be useful to pathologists in differentiating lesions, although a larger study should be evaluated to confirm these findings. Further, these features may be important as optical technologies are developed that make diagnoses in real time.