Performance estimation of diagnostic tests for cervical precancer based on fluorescence spectroscopy: effects of tissue type, sample size, population, and signal-to-noise ratio.

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.

Outcome and reproductive function after chemotherapy for ovarian dysgerminoma.

PURPOSE: To review the outcome for all patients with ovarian dysgerminoma treated at the M.D. Anderson Cancer Center who received bleomycin, etoposide, and cisplatin (BEP) and to assess the menstrual and reproductive function of those who received conservative treatment. PATIENTS AND METHODS: Clinical information was abstracted from the medical record. Patients completed a detailed questionnaire about menstrual and reproductive function; those who did not return the questionnaire were interviewed by telephone. RESULTS: Twenty-six patients were identified as having been treated with BEP chemotherapy for pure ovarian dysgerminoma from January 1984 to January 1998. Their median age was 19.5 years (range, 7 to 32 years). Sixteen patients underwent fertility-sparing surgery in the form of unilateral salpingo-oophorectomy. At a median follow-up time of 89 months, 25 (96%) of the 26 patients remained continuously disease-free. One patient apparently developed a second primary dysgerminoma in her remaining ovary after BEP and was clinically disease-free after further treatment. Of the 16 patients who underwent fertility-sparing surgery, one was lost to follow-up when she was pregnant, and one was still premenarchal. Of the remaining 14 patients, 10 (71%) maintained their normal menstrual function during and after chemotherapy, and 13 (93%) had returned to their prechemotherapy menstrual pattern at the time of the questionnaire. Five pregnancies have occurred thus far, and two patients have had difficulty conceiving. CONCLUSION: Most patients with metastatic dysgerminoma can expect cure with maintenance of normal reproductive function when treated with conservative surgery and BEP chemotherapy.

Screening for squamous intraepithelial lesions with fluorescence spectroscopy.

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.

Nuclear morphometry as an intermediate endpoint biomarker in chemoprevention of cervical carcinoma using alpha-difluoromethylornithine.

The use of nuclear morphometry as an intermediate endpoint biomarker is described in a Phase I, dose-seeking trial of chemoprevention of cervical cancer, using the agent alpha-difluoromethylornithine (DFMO). Thirty patients with grade III cervical intraepithelial neoplasia (CIN III) were enrolled, and these received daily doses of DFMO at 0.06-1.0 mg/m(2) for a period of 1 month. Fifteen patients were observed to have a complete or partial regressive response to the agent, as assessed by histopathology. No significant differences in cell feature measurements were found between responders and nonresponders in specimens obtained before treatment, indicating that it may be difficult to predict response on the basis of these measurements. In specimens collected after treatment, large differences in morphometric features were observed between responders and nonresponders, indicating a differential effect of DFMO. Significantly modulated features were considered in terms of their correlations with CIN grade, which was determined from an independent set of measurements from archival tissue. Differences between features were consistent with a deletion of cells with high grade nuclei in the responders, and with the persistence of a more heterogeneous population of high grade cells in the nonresponders. Based on an independent set of measurements from archival material, a morphometric index of progression was derived, yielding a quantitative measure of the degree of nuclear atypia in these lesions. When applied to this trial, the morphometric index was seen to be specifically and consistently decreased in responsive lesions, and unchanged in nonresponders. The study indicates that morphometric features fulfill the requirements for an intermediate endpoint biomarker of cervical cancer chemoprevention.

Fluorescence spectroscopy of the cervix: influence of acetic acid, cervical mucus, and vaginal medications.

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.

Decreased expression of retinoic acid receptors, transforming growth factor beta, involucrin, and cornifin in cervical intraepithelial neoplasia.

Cervical intraepithelial neoplasia (CIN) I, II, and III represent a spectrum of premalignant epithelial changes and are ideal targets for application of chemoprevention strategies. Intermediate end point biomarkers are increasingly being used as surrogate end points to monitor clinical chemoprevention trials. To identify potential biomarkers in cervical epithelium, we analyzed the expression of nuclear retinoic acid receptor (RAR) mRNA by in situ hybridization, involucrin, cornifin, and transforming growth factors (TGFs) beta1 and beta2 by immunohistochemistry in cervical specimens, which contained adjacent normal epithelium and CIN lesions from 52 patients. These biomarkers were expressed in all adjacent normal cervical epithelia, whereas all CIN lesions including CIN I, CIN II, and CIN III exhibited decreased expression of RAR-alpha by 55.8%, RAR-beta by 64.7%, RAR-gamma by 54.9%, involucrin by 80.8%, cornifin by 88.5%, TGF-beta1 by 89.7%, and TGF-beta2 by 85.7%. Viewed as a whole, these biomarkers were down-regulated in 100% of the CIN lesions. Because all of these biomarkers can be modulated in vitro by retinoids, they may serve as intermediate biomarkers for retinoid chemoprevention trials in the patients with CIN lesions.

Evaluation of cellular immune responses in rhesus monkeys subjected to adenovirus-mediated gene transfer into the cervix.

We reported previously that direct injection of a recombinant adenovirus (rAd), Ad5CMV-beta-gal, into the cervix of the rhesus monkey resulted in efficient beta-galactosidase expression in the cervix within 3 days. In these studies, we also observed the induction of anti-adenovirus (Ad)-specific immunoglobulin G responses after 22 days. In the continuation of evaluating the anti-Ad-specific immune responses resulting from this approach of gene targeting to the cervix, we measured the cellular immune responses. The introduction of Ad5CMV-beta-gal into the cervix by direct injection, but not by the abrasion technique, resulted in the induction of strong proliferative responses against extracts of cells infected with Ad5CMV-beta-gal but not against control uninfected cells. These responses were initially detected at 22 days postinjection and coincided with the abrogation of transgene expression. Significant levels of proliferative responses were maintained for < or =83 days. Multiple injections of rAds had no significant enhancing effect on either the level or longevity of the proliferative responses. At 3 days after the injection of Ad5CMV-beta-gal, when the transgene expression in the cervix was clearly evident, proliferative responses against the rAd were not detectable. However, the production of low but significant amounts of interleukin-10, a cytokine characteristic of T helper type 2 responses that promote humoral immune responses, was observed at the 3-day point in these animals. These results suggest that significant differences exist between the kinetics of transgene expression and the priming of specific host immune responses, and that these differences may be important for devising alternate strategies to improve techniques for Ad-mediated gene therapy of cervical cancer.

Prevention of ovarian cancer.

Chemoprevention trials with OCP and retinoids will be important to determine if these drugs are effective in selected populations. Further work will be critical to understanding the mechanism of action of OCP in preventing ovarian cancer and if this protective effect will be upheld in the high-risk population. Initial results in the Milan study are promising for the retinoids in prevention of ovarian cancer and will be used in chemoprevention trials both alone and in combination with OCP's to determine if there might be an additive effect with these drugs. As yet, little is known about preinvasive changes in the ovary to predict which women are at risk for developing ovarian cancer. We can now identify high-risk women by genetic counseling and testing, yet ultrasound and serum markers are the only modality available to evaluate these women. Research is focusing on developing ways of evaluating women, particularly those at high risk for ovarian cancer, to better understand the neoplastic process in the ovary and thus identify these women prior to their developing advanced ovarian cancer. Research is also focusing on understanding chemoprevention for ovarian cancer so that women can receive the optimal chemopreventive agent when diagnosed as high risk. Prognosis with advanced disease is so poor that early diagnosis and chemoprevention are the only methods at the current time to significantly improve survival in epithelial ovarian cancer.

Fluorescence spectroscopy for diagnosis of squamous intraepithelial lesions of the cervix.

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.

Human papillomavirus (HPV) DNA copy number is dependent on grade of cervical disease and HPV type.

The association between human papillomavirus (HPV) DNA copy number and cervical disease was investigated. Viral DNA copy number for the most common high-risk HPV types in cervical cancer (types 16, 18, 31, and 45) was determined in cervical cytobrush specimens from 149 women with high-grade cervical intraepithelial neoplasias (CIN II-CIN III), 176 with low-grade CIN (CIN I), and 270 with normal cytology. Quantitative, PCR-based fluorescent assays for each of the HPV genotypes and for the beta-globin gene were used. The amount of cellular DNA increased significantly with increasing disease; thus, HPV was expressed as copies per microgram of cellular DNA. The assay had a dynamic range of >10(7), allowing documentation for the first time of the wide range of HPV copy numbers seen in clinical specimens. Median HPV DNA copy number varied by more than 10(4) among the viral types. HPV16 was present in the highest copy number; over 55% of HPV16-positive samples contained more than 10(8) copies/microgram. Median copy number for HPV16 showed dramatic increases with increasing epithelial abnormality, an effect not seen with the other HPV types. HPV16 increased from a median of 2.2 x 10(7) in patients with normal cytology, to 4.1 x 10(7) in CIN I patients, to 1.3 x 10(9) copies/microgram in CIN II-III patients. Even when stratified by cervical disease and viral type, the range of viral DNA copies per microgram of cellular DNA was quite large, precluding setting a clinically significant cutoff value for "high" copy numbers predictive of disease. This study suggests that the clinical usefulness of HPV quantitation requires reassessment and is assay dependent.

Cervical fluorescence of normal women.

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.

Expression of human papillomavirus E7 mRNA in human oral and cervical neoplasia and cell lines.

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.

A case-control study of human papillomavirus and cervical squamous intraepithelial lesions (SIL) in Harris County, Texas: differences among racial/ethnic groups.

We conducted a case-control study of the association between SIL and HPV among whites (W), African Americans (AA), and Hispanics (H) in Harris County, Texas. Cases were identified at M.D. Anderson Cancer Center Colposcopy Clinic. Controls were identified among women obtaining routine Pap screening at two Harris County Health Department Clinics. HPV was detected by a PCR-based fluorescent assay. Dichotomous and polytomous logistic regression models were used to estimate adjusted odd ratios (AOR) and 95% confidence intervals (CI) for SIL among racial/ethnic groups and grade of disease. Prevalence of HPV infection was 64% in low grade SIL (LSIL), 84% in high grade SIL (HSIL), and 19% in controls. Risk of SIL was higher in H than in W and AA, AOR 29.5 (12.4-70.5), 15.3 (6.0-33.8), and 5.8 (2.6-12.6), respectively. Similarly, racial/ethnic differences were observed for both LSIL (AOR = 16.6, 7.7, and 4.3, respectively) and HSIL (AOR = 78.6, 34.6, and 14.2, respectively). Findings support the association between SIL and HPV and differences in the strength of the association with LSILs and HSILs. Data also suggest a higher risk for H and a lower risk for AA.

Noninvasive diagnosis of oral neoplasia based on fluorescence spectroscopy and native tissue autofluorescence.

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.

A randomized clinical trial of cryotherapy, laser vaporization, and loop electrosurgical excision for treatment of squamous intraepithelial lesions of the cervix.

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 chi2 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.

Development of a fiber optic probe to measure NIR Raman spectra of cervical tissue in vivo.

The goal of this study was to develop a compact fiber optic probe to measure near infrared Raman spectra of human cervical tissue in vivo for the clinical diagnosis of cervical precancers. A Raman spectrometer and fiber optic probe were designed, constructed and tested. The probe was first tested using standards with known Raman spectra, and then the probe was used to acquire Raman spectra from normal and precancerous cervical tissue in vivo. Raman spectra of cervical tissue could be acquired in vivo in 90 s using incident powers comparable to the threshold limit values for laser exposure of the skin. Although some silica signal obscured tissue Raman bands below 900 cm-1, Raman features from cervical tissue could clearly be discerned with an acceptable signal-to-noise ratio above 900 cm-1. The success of the Raman probe described here indicates that near infrared Raman spectra can be measured in vivo from cervical tissues. Increasing the power of the excitation source could reduce the integration time to below 20 s.

Near-infrared Raman spectroscopy for in vitro detection of cervical precancers.

In this study, we investigate the potential of near-infrared Raman spectroscopy to differentiate cervical precancers from normal tissues, inflammation and metaplasia and to differentially diagnose low-grade and high-grade precancers. Near infrared Raman spectra were measured from 36 biopsies from 18 patients in vitro. Detection algorithms were developed and evaluated relative to histopathologic examination. Algorithms based on empirically selected peak intensities, ratios of peak intensities and a combination of principal component analysis for data reduction and Fisher discriminant analysis for classification were investigated. Spectral peaks were tentatively identified from measured spectra of potential chromophores. Empirically selected normalized intensities can differentiate precancers from other tissues with an average sensitivity and specificity of 88 +/- 4% and 92 +/- 4%. Ratios of unnormalized intensities can differentiate precancers from other tissues with a sensitivity and specificity of 82% and 88% and high-grade from low-grade lesions with a sensitivity and specificity of 100%. Using multivariate methods, intensities at eight frequencies can be used to differentiate precancers from all other tissues with a sensitivity and specificity of 82% and 92% in an unbiased test. Raman algorithms can potentially separate benign abnormalities such as inflammation and metaplasia from precancers. Comparison of tissue spectra to published and measured chromophore spectra indicate that the most likely primary contributors to the tissue spectra are collagen, nucleic acids, phospholipids and glucose 1-phosphate. These results suggest that near-infrared Raman spectroscopy can be used for cervical precancer diagnosis and may be able to accurately separate samples with inflammation and metaplasia from precancer.

Epidermal growth factor receptor expression in cervical intraepithelial neoplasia and its modulation during an alpha-difluoromethylornithine chemoprevention trial.

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.

Phase I dose de-escalation trial of alpha-difluoromethylornithine in patients with grade 3 cervical intraepithelial neoplasia.

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.

Colposcopy for the diagnosis of squamous intraepithelial lesions: a meta-analysis.

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.