BACKGROUND:This study reviews the experience at the University of Texas M.D. Anderson Cancer Center with elderly patients treated for advanced epithelial ovarian cancer with cisplatin-based combination chemotherapy.METHODS:From 1978 through 1988, 215 patients with Stage III or IV, Grade 2 or 3 epithelial ovarian cancer were entered onto one of three consecutive trials involving cisplatin-based combination chemotherapy. The treatment plans were as follows: Trial 1: 12 cycles of cisplatin-melphalan (75 patients); Trial 2: 12 cycles of cisplatin-cyclophosphamide (49 patients); and Trial 3: 6 cycles of cisplatin-cyclophosphamide (91 patients). End-points of analysis included the effect of age on other prognostic factors, clinical response rate, surgical response rate, toxicity, progression-free survival, and survival.RESULTS:Of the entire study group, 57 patients (27%) were 65 years of age and older at diagnosis. There was no effect of age on FIGO stage or histologic grade distribution, but 61% of patients younger than 65 years of age had residual tumors less than or equal to 2 cm compared with only 33% of patients 65 years of age and older (P = 0.00027). There were no differences between the two age groups with respect to response rates, recurrence rate after negative second-look surgery, toxicity, or progression-free survival. Patients in the younger than 65 years of age group, however, had a significantly longer median survival time than those 65 years of age and older (30 versus 19 months, respectively) (P = 0.0038).CONCLUSIONS:This analysis suggests that elderly patients are more likely to begin chemotherapy with bulky residual disease and to have a significantly shorter survival time than their younger counterparts.

Background and Objective: Fluorescence spectroscopy has been shown to provide information useful in the detection of cervical dysplasia. The goal of this study was to determine if substances found on the cervix such as acetic acid, mucus, and vaginal medications can influence the fluorescence in the spectral region useful for discriminating normal cervical tissue from abnormal tissue.Study Design/Materials and Methods: Fluorescence spectra were collected at 337 nm excitation from the cervix in vivo both before and after application of acetic acid; the data were analyzed to identify the effects of the acetic acid on the spectra. Cervical mucus was acquired from patients referred for colposcopy and frozen until measurements were taken. Fluorescence excitation-emission matrices (EEMs) were measured for the mucus samples. Additionally, the transmission spectra of mucus were measured to determine if its absorption could influence the fluorescence signal measured from the tissue, EEMs were measured for samples of commonly prescribed vaginal medications, All EEMs were compared to those of cervical biopsies,Results: Acetic acid introduces changes in both the lineshape and intensity of the spectra, On average, the changes are more significant in spectra of abnormal tissue, Cervical mucus was found to have no significant absorption bands, but the measured fluorescence was approximately the same order of magnitude as that measured from the cervix in vitro, Most medications exhibited significant fluorescence in the spectral region of diagnostic interest for the cervix.Conclusions: Acetic acid appears to increase the differences in fluorescence emission spectra of normal and pre-cancerous cervical tissues; thus, its use is beneficial, The presence of cervical mucus can possibly interfere with the collection of fluorescence spectra for tissue classification. Patients should not use vaginal preparations during the 48 hours prior to tissue fluorescence measurements. (C) 1999 Wiley-Liss, Inc.

Fluorescence spectroscopy may provide a cost-effective tool to improve precancer detection. We describe a method to estimate the diagnostic performance of classifiers based on optical spectra, and to explore the sensitivity of these estimations to factors affecting spectrometer cost. Fluorescence spectra were obtained at three excitation wavelengths in 92 patients with an abnormal Papanicolaou smear and 51 patients with no history of an abnormal smear. Bayesian classification rules were developed and evaluated at multiple misclassification costs. We explored the sensitivity of classifier performance to variations in tissue type, sample size, tested population, signal to noise ratio (SNR), and number of excitation and emission wavelengths. Sensitivity and specificity could be evaluated within +/- 7%. Minimal decrease in diagnostic performance is observed as SNR is reduced to 15, the number of excitation-emission wavelength combinations is reduced to 15 or the number of excitation wavelengths is reduced to one. Diagnostic performance is compromised when ultraviolet excitation is not included. Significant spectrometer cost reduction is possible without compromising diagnostic ability. Decision-analytic methods can be used to rate designs based on incremental cost-effectiveness.

OBJECTIVE:To calculate receiver operating characteristic (ROC) curves for fluorescence spectroscopy in order to measure its performance in the diagnosis of squamous intraepithelial lesions (SILs) and to compare these curves with those for other diagnostic methods: colposcopy, cervicography, speculoscopy, Papanicolaou smear screening, and human papillomavirus (HPV) testing. DATA SOURCES:Data from our previous clinical study were used to calculate ROC curves for fluorescence spectroscopy. Curves for other techniques were calculated from other investigators' reports. To identify these, a MEDLINE search for articles published from 1966 to 1996 was carried out, using the search terms "colposcopy," "cervicoscopy," "cervicography," "speculoscopy," "Papanicolaou smear," "HPV testing," "fluorescence spectroscopy," and "polar probe" in conjunction with the terms "diagnosis," "positive predictive value," "negative predictive value," and "receiver operating characteristic curve." METHODS OF STUDY SELECTION:We found 270 articles, from which articles were selected if they reported results of studies involving high-disease-prevalence populations, reported findings of studies in which colposcopically directed biopsy was the criterion standard, and included sufficient data for recalculation of the reported sensitivities and specificities. TABULATION, INTEGRATION, AND RESULTS:We calculated ROC curves for fluorescence spectroscopy using Bayesian and neural net algorithms. A meta-analytic approach was used to calculate ROC curves for the other techniques. Areas under the curves were calculated. Fluorescence spectroscopy using the neural net algorithm had the highest area under the ROC curve, followed by fluorescence spectroscopy using the Bayesian algorithm, followed by colposcopy, the standard diagnostic technique. Cervicography, Papanicolaou smear screening, and HPV testing performed comparably with each other but not as well as fluorescence spectroscopy and colposcopy. CONCLUSION:Fluorescence spectroscopy performs better than colposcopy and other techniques in the diagnosis of SILs. Because it also permits real-time diagnosis and has the potential of being used by inexperienced health care personnel, this technology holds bright promise.

Objective: To compare cryotherapy, laser vaporization, and loop electrical excision for treatment of squamous intraepithelial lesions (SILs).Methods: Women at least 18 years old with biopsy-proven SIL, negative pregnancy tests, negative findings on endocervical curettage, satisfactory colposcopy examinations, and congruent Papanicolaou smear and biopsy results were assigned randomly to treatment after stratification by SIL grade, endocervical gland involvement, and lesion size; they were evaluated 1, 4, 8, 12, 16, 20, and 24 months after treatment. Data were analyzed using chi(2) statistics, logistic regression analysis, and the Cox proportional hazards model.Results: Of 498 patients assigned, 108 were excluded (most because of inadequate follow-up), leaving 390 (139 cryotherapy, 121 laser vaporization, 130 loop excision) for analysis. All were followed 6-37 months (mean 16). There were no statistically significant differences in complications, persistence (disease present less than 6 months after treatment), or recurrence (disease present more than 6 months after treatment). Risk of persistent disease was higher among women with large lesions (risk ratio [RR], 18.9; 95% confidence interval [CI], 3.2, 110.6). Recurrence risk was higher among women aged 30 years and older (RR, 2.1; 95% CI, 1.2, 4.3), those with human papillomavirus type 16 or 18 (RR, 2.1; 95% CI, 1.1, 4.0), and those who had had prior treatment (RR, 2.1; 95% CI, 1.1, 3.9).Conclusion: The data support a high success rate with all three modalities. No significant difference in success rates was observed between the three treatments in our population. Additional attention and research should be directed toward the higher risk patients identified above. (Obstet Gynecol 1998;92:737-44. (C) 1998 by The American College of Obstetricians and Gynecologists.)

Background and Objective Cervical tissue fluorescence spectra have previously been measured in vivo in women with a recent abnormal Papanicolaou smear. Diagnostic algorithms have been developed to diagnose squamous intraepithelial lesions (SILs) based on these fluorescence emission spectra. However, algorithms have not been tested in women with no history of cervical neoplasia. Study Design/Materials and Methods Cervical fluorescence was measured from 54 women with no history of cervical dysplasia, and the spectra were compared to those from colposcopically normal sites in women with suspected dysplasia. Representative spectra from each group were compared and a two-sided, unpaired Student's t-test was performed to compare mean principal component scores used in previously published diagnostic algorithms. The ability of previously reported diagnostic algorithms to classify these samples as normal tissue was also assessed. Results At the 0.05 level of significance, the mean scores of 4 of the 7 important principal components were statistically different for the two populations. However, when the data collected from volunteers in this study were preprocessed in the appropriate manner and the algorithms were applied, more normal samples were correctly classified than in the previous clinical study in which these algorithms were developed. Conclusion Previously reported algorithms can accurately classify tissue type based on spectra from women with and without a history of cervical neoplasia. Lasers Surg. Med. 24:29–37, 1999. © 1999 Wiley-Liss, Inc.

Human papillomaviruses (HPVs) have been strongly linked to progression of human cancers, such as cervical and oral cancers. Two HPV oncoproteins, E6 and E7, can inhibit the tumor suppressor proteins, p53 and pRB, respectively, resulting in a deregulation of the cell cycle. In order to further test the significance of HPV expression in oral and cervical carcinogenesis, we analyzed HPV E7 mRNA in oral and cervical neoplasia and cell lines by reverse transcriptase-polymerase chain reaction (RT-PCR). We found that HPV E7 mRNA was present in 90% of patients with oral neoplasia and 100% of patients with cervical neoplasia. Quantitative RT-PCR and western blot analysis on both transformed cervical and oral epithelial cell lines demonstrated that the mRNA level of HPV-16 E7 corresponded to E7 protein level, suggesting that HPV oncogene expression is primarily regulated at the transcriptional or post-transcription level. The potential clinical application of quantitative RT-PCR for HPV E7 mRNA expression in cancer screening and treatment evaluation requires further investigation.

Objective: To evaluate the accuracy of fluorescence spectroscopy in screening for squamous intraepithelial lesions (SILs) and to compare its performance with that of Papanicolaou smear screening, colposcopy, cervicoscopy, cervicography, and human papillomavirus (HPV) testing.Data Sources: Receiver operating characteristic (ROC) curve analysis was used to analyze performance by fluorescence spectroscopy (primary data) and other methods (secondary data).Methods of Study Selection: In our search, 275 articles were identified in MEDLINE (1966-1996). Articles were included if the investigators had studied a population in whom low disease prevalence was expected; used either Papanicolaou smear screening and colposcopy or colposcopically directed biopsy as a standard against which the screening technique was measured, and included enough data for recalculation of reported sensitivities and specificities.Tabulation, Integration, and Results: Receiver operating characteristic curves for fluorescence spectroscopy were calculated using a Bayesian algorithm, and ROC curves for the other screening methods were constructed using metaanalytic techniques. Areas under the ROC curves and Q points were calculated. Screening colposcopy had the highest area under the curve (0.95), followed by screening cervicography (0.90), HPV testing (0.88), cervicoscopy (0.85), fluorescence spectroscopy (0.76), and Papanicolaou smear screening (0.70).Conclusion: In terms of screening for SILs, fluorescence spectroscopy performed better than the standard technique, Papanicolaou smear screening, and less well than screening colposcopy, cervicography, HPV testing, and cervicoscopy. The promise of this research technique warrants further investigation. (Obstet Gynecol 1999;94:889-96. (C) 1999 by The American College of Obstetricians and Gynecologists.).

Objective: To compare five strategies for the diagnosis and treatment of cervical squamous intraepithelial lesions (SILs), including those that incorporate colposcopy and a new technology, fluorescence spectroscopy.Methods: On the basis of a health care perspective, we performed a cost-effectiveness analysis using a decision-analytic model for the diagnosis and management of SILs. We compared the five strategies based on the expected costs and number of cases that were treated appropriately, missed, treated inappropriately, and appropriately not treated in a hypothetical cohort of 100 patients referred after an abnormal Papanicolaou smear. Data on prevalence and operating characteristics were derived from the medical literature. Costs were adjusted from hospital charge data.Results: A see-and-treat strategy based on fluorescence spectroscopy was the least expensive but least effective strategy, costing $160,479 to detect 31.55 cases of cervical precancer accurately in 100 patients. The most expensive strategy was colposcopically directed biopsy, at $311,808 to find 45.78 cases; however, when both tests were used in a see-and-treat modality, slightly more cases were found (46.05) at a lower cost ($285,133). Other strategies were dominated in the base case. The incremental cost-effectiveness of the joint strategy compared with the spectroscopy-only strategy was $8596 per case of cervical precancer detected. Sensitivity analysis showed that the analysis was sensitive to the cost of the new technology of fluorescence spectroscopy.Conclusion: Fluorescence spectroscopy should be considered an important innovation in the diagnosis of SILs as demonstrated by its efficacy and economic advantages. (C) 1998 by The American College of Obstetricians and Gynecologists.).

This study evaluates the potential of near-infrared Raman spectroscopy for in vivo detection of squamous dysplasia, a precursor to cervical cancer. A pilot clinical trial was carried out at three clinical sites. Raman spectra were measured from one colposcopically normal and one abnormal area of the cervix. These sites were then biopsied and submitted for routine histologic analysis. Twentyfour evaluable measurements were made in vivo in 13 patients. Cervical tissue Raman spectra contain peaks in the vicinity of 1070, 1180, 1195, 1210, 1245, 1330, 1400, 1454, 1505, 1555, 1656, and 1760 cm −1 . The ratio of intensities at 1454 to 1656 cm −1 is greater for squamous dysplasia than all other tissue types, while the ratio of intensities at 1330 to 1454 cm −1 is lower for samples with squamous dysplasia than all other tissue types. A simple algorithm based on these two intensity ratios separates high-grade squamous dysplasia from all others, misclassifying only one sample. Spectra measured in vivo resemble those measured in vitro. Cervical epithelial cells may contribute to tissue spectra at 1330 cm −1 , a region associated with DNA. In contrast, epithelial cells probably do not contribute to tissue spectra at 1454 cm −1 , a region associated with collagen and phospholipids.

Chemoprevention trials designed to prevent progression to invasive cervical cancer will benefit from the identification of biomarkers that assess the risk of developing tumors, predict likelihood of response to treatment, and measure biological response to intervention. The purpose of this study was to examine expression of epidermal growth factor receptor (EGFR) as a marker for progression of cervical intraepithelial neoplasia (CIN) and as a surrogate end point biomarker in a chemoprevention trial with alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase. To evaluate quantitative and spatial changes in EGFR expression during cervical tumorigenesis, paraffin sections from 42 archival cervical cone biopsies, each containing multiple stages of CIN, were immunohistochemically stained for EGFR, and the level and spatial expression of EGFR were quantitated by image analysis. In the progression from normal epithelium to CIN 1 to CIN 2 to CIN 3 to invasive cancer, EGFR expression showed two types of changes. Normal control epithelium showed EGFR expression predominantly confined to the basal layer, while histologically normal epithelium in specimens containing CIN showed relatively increased EGFR expression in the basal layer and the extension of EGFR expression away from the basal layer. The total EGFR relative staining intensity (RSI) of epithelium increased with the degree of CIN, predominantly due to a progressive expansion of EGFR-expressing cells away from the basal layer rather than an increase in the level of EGFR expression per cell. To determine whether EGFR expression would be modulated by a 1-month chemopreventive intervention with DFMO, pretreatment and posttreatment cervical biopsy specimens from 25 patients (22 evaluable) were examined for EGFR expression. Although the overall levels of EGFR expression were not modulated in either histological responders or nonresponders, responders showed a prominent down-regulation of EGFR expression away from the basal layer after DFMO treatment. Interestingly, pretreatment EGFR expression levels predicted for DFMO response [i.e., eight responses (72.7%) for 11 cases with RSI levels below 0.35 versus one response (9.1%) for 11 cases with RSI levels above 0.35 (P < 0.01)]. These results suggest that CIN progression is associated with a spatial dysregulation of EGFR expression that can be reversed by DFMO treatment, especially in patients whose pretreatment CIN 3 lesions exhibit relatively low EGFR expression.

Objective: To quantify by meta-analysis the performance of colposcopy to set a standard against which new technologies can be compared.Data Sources: MEDLINE was searched for articles on colposcopy for diagnosis of squamous intraepithelial lesions (SIL). The search selected articles from 1960 to 1996 combining the key word "colposcopy" with key words "diagnosis," "positive predictive value," "negative predictive value," "likelihood ratio," and "receiver operating characteristic (ROC) curve."Methods of Study Selection: Articles were selected if the authors studied a population of patients with abnormal screening Papanicolaou smears and presented raw data showing for each cervical lesion type the number of patients judged positive and negative by colposcopic impression versus the standard of colposcopic biopsy results. Nine of 86 studies met these criteria.Tabulation, Integration, and Results: Biopsies had been categorized as normal, atypia, cervical intraepithelial neoplasia (CIN) I, CIN II, CIN III, carcinoma in situ, and invasive cancer; we recalculated performance measures using the Bethesda system. Overall sensitivity, specificity, likelihood ratios, ROC curves, and the corresponding areas under the curves were calculated. The average weighted sensitivity of diagnostic colposcopy for the threshold normal compared with all cervix abnormalities (atypia, low-grade SIL, high-grade SIL, cancer) was 96% and the average weighted specificity 48%. For the threshold normal cervix and low-grade SIL compared with high-grade SIL and cancer, average weighted sensitivity was 85% and average weighted specificity 69%. Likelihood ratios generated small but important changes in probability for distinguishing normal cervix and low-grade SIL from high-grade SIL and cancer. Areas under the ROC curve were 0.80 for the threshold normal cervix compared with all abnormalities and 0.82 for the threshold normal cervix and low-grade SIL compared with high-grade SIL and cancer.Conclusion: Colposcopy compares favorably with other medical diagnostic tests in terms of sensitivity, specificity, and area under the ROC curve. New diagnostic methods for the cervix can be compared with colposcopy using these quantified values. (C) 1998 by The American College of Obstetricians and Gynecologists.

alpha-Difluoromethylornithine (DFMO) is a suicide inhibitor of ornithine decarboxylase and potent antiproliferative chemopreventive agent. We conducted a dose de-escalation Phase I trial of DFMO in patients with grade 3 cervical intraepithelial neoplasia to determine an optimal dose of DFMO using ornithine decarboxylase activity and polyamine modulation as surrogate biomarkers and to evaluate its toxicity. Thirty patients with biopsy-confirmed grade 3 cervical intraepithelial neoplasia were assigned sequentially to one of five DFMO doses (1.000, 0.500, 0.250, 0.125, or 0.060 g/m2) given daily for 31 days. One patient was excluded from analysis for protocol violation. Polyamine levels were assessed in cervical tissue, plasma, and RBCs. Tissue and blood samples were obtained before and after treatment with DFMO. All patients underwent loop excision of the cervix at the end of the study for complete histological evaluation and definitive treatment of the premalignant condition. No major clinical toxicity was observed at any DFMO dose. A reduction in tissue spermidine to spermine (SPD:SPM) ratio and an increase in plasma arginine levels were observed among patients receiving 1.000 g/m2/day (P < 0.05). A nonsignificant reduction in SPD:SPM ratio was also observed in the 0.500 g/m2/day dose group, and a nonsignificant increase in plasma arginine level was observed down to the 0.125 g/m2/day dose level. There was no evidence of modulation of other polyamines or precursors. Fifteen patients experienced a complete (5 patients) or partial (10 patients) histological response. In conclusion, DFMO was well tolerated and significantly modulated tissue SPD:SPM ratio and plasma arginine level at the dose of 1.000 g/m2/day. To clarify whether DFMO has activity at lower doses, these results will be tested in a three-armed double-blinded Phase II study using placebo and DFMO doses of 0.500 and 0.125 g/m2/day.

Recent clinical trials have demonstrated the potential of fluorescence spectroscopy for in vivo diagnosis of pathology. There is significant potential to reduce the cost and complexity of instrumentation to measure tissue spectra; however, careful analysis is required to maximize performance and minimize cost. One measure of performance is the signal-to-noise ratio (SNR) of the resulting data. This paper describes a method to predict the SNR of a given optical design for a particular tissue application. In order to calculate the expected SNR, two pieces of information are required: (1) the throughput and inherent noise of the system and (2) a quantitative relationship between the illumination energy and the resulting tissue fluorescence available for collection, which we define as the tissue fluorescence efficiency (FE). We present a method to calculate the fluorescence efficiency of tissue from in vivo measurements of tissue fluorescence. We report FE measurements of the normal and precancerous human cervix in vivo at 337, 380, and 460 nm excitation. We also present and evaluate a method to estimate the throughput and noise of various spectrometers and predict the expected SNR for tissue spectra by using the measured tissue FE. For squamous cervical tissue, as the degree of the disease increases, FE decreases, and as the excitation wavelength increases, FE decreases. Cervical tissue FE varies more than two orders of magnitude, depending on the tissue type and on the excitation wavelength used. Our SNR calculations, based on measured values of tissue FE, demonstrate agreement within a factor of 1.3 of the measured SNR on average. This method can be used to estimate the performance of different spectrometer designs for clinical use.

OBJECTIVE:To evaluate the clinical potential of fluorescence spectroscopy (a noninvasive technique for assessing the chemical and morphologic composition of tissue) for in vivo detection of oral cavity neoplasia.DESIGN:A fluorescence spectroscopy system recorded spectra from oral cavity sites in 8 healthy volunteers and in 15 patients with premalignant or malignant oral cavity lesions at 337-, 365-, and 410-nm excitation wavelengths in the emission range of 350 to 700 nm. Fluorescence peak intensities and spectral line shapes were compared and diagnostic algorithms were developed to distinguish normal sites from abnormal sites.SETTING:The head and neck cancer clinic at a tertiary referral center in Houston, Tex.RESULTS:Differences were found in spectra from normal, dysplastic, and malignant oral mucosa. The fluorescence intensity of normal mucosa was greater than that of abnormal areas. In addition, the ratio of red region (635-nm) to blue region (455-490-nm) intensities was greater in abnormal areas. Diagnostic discrimination was achieved when test site spectra were compared with spectra from a normal site in the same patient. One diagnostic algorithm based on spectra at 337 nm gave a sensitivity of 88% and a specificity of 100%.CONCLUSIONS:Consistent differences exist between the fluorescence spectra of abnormal and normal oral mucosa. Therefore, fluorescence spectroscopy has the potential to improve the noninvasive diagnosis of oral cavity neoplasia. Further studies will better define the role of this technique in the detection of premalignant and early oral cancer lesions.

Cervical cancer remains a significant health problem. New strategies based on the molecular aspects of cervical carcinogenesis are needed. Chemoprevention represents a novel strategy for cervical cancer prevention. Our group plans phase I and II trials using alpha-difluoromethylornithine, a suicide inhibitor of ornithine decarboxylase and potent antiproliferative chemopreventive agent. We conducted a study to identify which polyamines in tissue could best serve as surrogate endpoint biomarkers for future trials. Thirty patients with biopsy-proven cervical intraepithelial neoplasia grade 3 underwent colposcopically directed biopsies of normal and abnormal areas of the uterine cervix for analysis of polyamine synthesis biomarkers. Statistically significant differences were found in the ornithine decarboxylase value and the spermidine:spermine ratio between normal and abnormal areas of the cervix. In general, the ranges in measurements varied widely. Differences in polyamine synthesis biomarkers between colposcopically normal and abnormal areas can be demonstrated. However, studies using polyamine synthesis biomarkers in the cervix would require large numbers of patients to achieve significance.

Fluorescence spectroscopy is a promising tool for use in the diagnosis of disease in human tissue. However, few published reports have evaluated the safety of this technique, despite the fact that many spectroscopic systems use UV illumination. This study determined the relative risk associated with light exposure from spectroscopic systems compared with the traditional light sources that are used to illuminate tissue and direct biopsies. We compared spectroscopic detection systems for the cervix to the colposcope, a low-power microscope routinely used to illuminate the cervix, which does not cause any known photochemical damage. We measured the average spectral irradiance (W/[cm(2)nm]) and the average tissue exposure time during a diagnostic colposcopy examination. To quantify the relative risks, we multiplied illumination spectra by several action spectra from the literature and compared the areas under the curves corresponding to each procedure. The risk associated with the average power colposcope served as our basis for comparison, We conclude that the risks of illumination using spectroscopic systems are lower than or comparable to those already encountered in routine diagnostic procedures such as colposcopy with an average power colposcope. Spectroscopic examination can be associated with a somewhat higher risk than a colposcopy with the lowest power colposcope or a shorter than average colposcopy. The analysis presented can be repeated to estimate the magnitude of risks associated with other spectroscopic diagnostic devices.

BACKGROUNDThe inhibitory effects of N-(4-hydroxyphenyl)retinamide (4HPR) on tumorigenesis and tumor growth may result from its ability to induce apoptosis (programmed cell death). Since antioxidants inhibit 4HPR-induced apoptosis, experiments were planned to determine whether the levels of reactive oxygen species increase in cells undergoing apoptosis after exposure to 4HPR.METHODSCells of the human cervical carcinoma cell line C33A and normal human cervical epithelial cells were treated with 4HPR and analyzed for survival, induction of apoptosis, generation of reactive oxygen species, and expression of the apoptosis-related proteins Bcl-2 and Bax.RESULTSTreatment with 4HPR decreased C33A cell number by inducing apoptosis in a time- and dose-dependent fashion. DNA fragmentation typical of apoptosis was observed in cells exposed to 4HPR at concentrations of 3 microM or higher for 6-24 hours. The generation of reactive oxygen species was enhanced by 1.85-fold to 4.5-fold after a 1.5-hour treatment with 0.4-10 microM 4HPR. Pyrrolidine dithiocarbamate, an oxygen radical scavenger, suppressed the rate of generation of reactive oxygen species and inhibited 4HPR-induced apoptosis. 4HPR failed to modulate cellular levels of the Bcl-2 and Bax proteins. N-(4-Methoxyphenyl)retinamide, the major 4HPR metabolite, and several other retinoids that bind to nuclear retinoic acid receptors or retinoid X receptors failed to enhance the generation of reactive oxygen species and to induce apoptosis. 4HPR was much less effective in generating reactive oxygen species and in inducing apoptosis in normal human cervical epithelial cells than in C33A cervical carcinoma cells.CONCLUSIONSEnhancement of the generation of reactive oxygen species may be involved in apoptotic pathway induction by 4HPR.

BACKGROUND. Although hormone receptor status is an important prognostic indicator in adenocarcinoma of the breast and the endometrium, few studies have investigated the expression and clinical significance of estrogen receptor (ER) and progesterone receptor (PgR) in adenocarcinoma of the cervix. METHODS. ER and PgR expression were determined using an immunohistochemical method in 84 cervical adenocarcinomas. Clinical features and outcome were determined by chart review. RESULTS, ER was identified in 17 of the 84 cases (20%). ER positivity was most frequently detected in mucinous adenocarcinoma of the endocervical type (in 11 of 48 cases) and endometrioid adenocarcinoma (in 4 of 10 cases). PgR was identified in 23 of the 84 cases (27%). PgR positivity was also most frequently detected in mucinous adenocarcinoma of the endocervical type (in 15 of 48 cases) and endometrioid adenocarcinoma (in 6 of 10 cases). Mucinous adenocarcinoma of the intestinal type (five cases), glassy cell carcinoma (two cases), and clear cell adenocarcinoma (two cases) were uniformly negative for both ER and PgR. No association was detected between International Federation of Gynecology and Obstetrics stage and receptor status, but there was a somewhat lower frequency of ER positivity in poorly differentiated tumors (P = 0.07). No association was detected between PgR status and disease free survival. Similarly, no association between ER status and overall survival was observed. Although ER positive tumors may be associated with longer disease free survival than ER negative tumors, this difference did not reach statistical significance in this study (P = 0.06); CONCLUSIONS, ER and PgR positivity were found in 20% and 27%, respectively, of primary cervical adenocarcinomas. However, receptor status was not significantly associated with either overall survival or disease free survival. (C) 1997 American Cancer Society.

Cervical intraepithelial neoplasia grade 3 (CIN 3) is considered a high-risk precursor of invasive cervical cancer. alpha-Difluoromethylornithine (DFMO) is a promising antiproliferative chemopreventive agent. The purpose of this study was to evaluate image cytometric measurement of nuclear DNA (ICM-DNA) as a surrogate end point biomarker (SEB) in a Phase I trial of DFMO for CIN. Thirty patients with CIN 3 were treated with DFMO at five doses, ranging from 0.0625 to 1.0 g/m2/day, for 1 month. Half of the patients had histological responses. Twenty-five pre- and posttreatment cervical biopsy specimens (from 11 responders and 14 nonresponders) were available for this analysis. ICM-DNA was performed on 4-micron sections cut from formalin-fixed tissue blocks and stained with a thionin-SO2 Feulgen reaction. ICM-DNAs for each case were expressed as normalized measurements (against the nuclear modal absorbance of lymphocytes) of the absorbance of each cell of interest and were presented in bar histograms. The mean normalized summed absorbance (sigma ODn) was obtained as a mean histogram of the cell population of interest. Nineteen (76%) of 25 patients had a significant decrease in sigma ODn after DFMO treatment. Posttreatment values were significantly lower than pretreatment values in a paired analysis, and responders had significantly lower values than nonresponders. Analyses of different ICM-DNA references, including percentile values of sigma ODn distribution, DNA malignancy grade, and 5c exceeding rate, showed a decrease of mean sigma ODn during DFMO treatment. In addition, the summed posttreatment sigma ODn histograms also showed progressively shorter right shoulders compared with pretreatment histograms in both responders and nonresponders. We concluded that the modulation of sigma ODn reflected the chemoprevention effect of DFMO even before morphological changes appeared, and thus, ICM-DNA may be useful as a SEB in chemoprevention trials of DFMO. Additional reasons for using ICM-DNA as a SEB are the relative simplicity of its use, the high accuracy of the results, the low cost of the reagents, the ability to use small tissue samples, and the objectivity and reproducibility of the procedure.