Lymphoproliferative responses to human papillomavirus (HPV) type 16 proteins E6 and E7: outcome of HPV infection and associated neoplasia.

BACKGROUND: Infection with human papillomavirus (HPV) type 16 (HPV16) is a major cause of high-grade cervical intraepithelial neoplasia (CIN). Experiments were planned to evaluate the role of cell-mediated immunity (e.g., lymphocyte proliferation) against HPV in the natural history of HPV-associated neoplasia and to identify antigenic sequences of the HPV16 proteins E6 and E7 against which an immune response may confer protection. METHODS: Forty-nine women with abnormal cervical cytology and biopsy-confirmed CIN were followed through one or more clinic visits. Lymphoproliferative responses of peripheral blood mononuclear cells to HPV16 E6 and E7 peptides were assessed in long-term (3-week) cultures. HPV DNA was detected in cervicovaginal lavage by means of polymerase chain reaction and Southern blotting. Disease status was determined by cervical cytologic examination and colposcopy. Reported P values are two-sided. RESULTS: Subjects with positive lymphoproliferative responses to E6 and/or E7 peptides were more likely to be HPV negative at the same clinic visit than were nonresponders (P = .039). Subjects who were negative for HPV and those with a low viral load were more likely to be responders than were those with a high viral load (P for trend = .037). Responses to N-terminal E6 peptide 369 were associated with absence of HPV infection at the same clinic visit (P = .015). Subjects with positive responses to E6 or E7 peptides at one clinic visit were 4.4 times more likely to be HPV negative at the next visit than were nonresponders (P = .142). Responses to E6 peptide 369 and/or E7 C-terminal peptide 109 were associated with an absence of HPV infection (P = .02 for both) and an absence of CIN (P = .04 and .02, respectively) at the next visit. CONCLUSIONS: Lymphoproliferative responses to specific HPV16 E6 and E7 peptides appear to be associated with the clearance of HPV infection and the regression of CIN.

Cellular binding proteins for vitamin A in human carcinomas and in normal tissues.

Blinded analyses of the concentrations of binding proteins for retinol and retinoic acid (CRABP) in homogenates of cancer and normal tissue aliquots obtained from human cervix, endometrium, ovary, breast, and lung were carried out by the sucrose gradient ultracentrifugation technique. In carcinomas of the cervix and endometrium, CRABP mean values of 50.4 and 123.2 pmol/g tissue, respectively were detected. Such concentrations represent a 3- and 4-fold increase over the mean values of CRABP in the normal cervix (16.9 pmol/g) and normal endometrium (30.8 pmol/g), respectively. In carcinomas of the ovary, the mean CRABP level was 128.6 pmol/g compared to the maximal mean value of less than or equal to 0.46 pmol/g in the normal ovary. Elevated levels of CRABP were also found in breast and lung carcinomas compared to the amounts detected in the same patient in normal tissue aliquots of the same organ. The differences between CRABP concentrations in cervical, endometrial, ovarian, and breast carcinomas and those in normal tissue are statistically significant. In contrast, cellular retinol-binding protein concentrations were reduced in the endometrial, ovarian, breast, and lung carcinomas compared to normal tissues. There were no significant differences between the log-mean concentrations of cellular retinol-binding proteins in the cytosols from tissue aliquots of carcinoma of the cervix and those in the cytosols from tissue aliquots of normal cervix.

Cellular binding proteins for vitamin A in the normal human uterine cervix and in dysplasias.

Cellular retinol-binding protein (CRBP) and cellular retinoic acid-binding protein are present in the cytosol of normal human uterine cervical tissues, as detected by ultracentrifugation analysis. Both binding proteins have characteristically high specificity for their respective ligands. In sucrose gradients, both proteins sediment in the 2S region and are of similar molecular weight (M.W. approximately 14,000). In blind analyses of cervical biopsies, obtained under direct vision by colposcopy of normal women (control) or from patients histopathologically diagnosed to have dysplasias or carcinoma in situ (study group), CRBP was not detectable by sucrose gradient analysis in 78.8% of the 33 abnormal biopsies, compared to 23.5% of the 34 controls. This difference was statistically significant (p less than 0.005). In biopsies in which CRBP was detected, the mean levels were 2.76 and 0.72 pmol/mg protein in the cytosol for the control and study groups, respectively. In some subjects from each group, cellular retinoic acid-binding protein but not CRBP was detected in the biopsied tissue. The presence and role of these binding proteins in vitamin A metabolism, epithelial maturation and differentiation in cervical dysplasias, and in situ lesions remain to be investigated.

Urinary cyclic nucleotides and the cytopathology of human uterine cervical dysplasias.

Blinded urinary assays for cyclic guanosine 3':5'-monophosphate (cGMP) and cyclic adenosine 3':5'-monophosphate (cAMP) were performed on 49 subjects with documented abnormal cervical cytology and 21 control subjects with normal cytology. A significant difference in the mean cGMP:cAMP ratios between the case and control groups was found. A significantly greater proportion of women with cytological abnormalities had a cGMP:cAMP ratio above the 0.2 level (p less than 0.001). Cases treated surgically for severe dysplasia or carcinoma in situ of the cervix revealed a significant postsurgical fall in the cGMP:cAMP ratios (p less than 0.025). The possibility of utilizing urinary ratios of cyclic nucleotides as an objective index in the detection, monitoring of progression, and therapy of preneoplastic cervical lesions is discussed.

Plasma levels of beta-carotene, lycopene, canthaxanthin, retinol, and alpha- and tau-tocopherol in cervical intraepithelial neoplasia and cancer.

Epidemiological studies continue to identify an association of dietary antioxidant micronutrients in cancer prevention. A number of case-control and cohort studies have demonstrated a relationship between high intake of foods rich in carotenoids, tocopherols, and vitamin C with a reduced risk of certain human malignancies. The purpose of this study was to investigate the comparative plasma levels of a profile of known dietary antioxidants, namely, beta-carotene, lycopene, canthaxanthin, retinol, alpha-tocopherol, and tau-tocopherol. The target population was women with a histopathological diagnosis of cervical intraepithelial neoplasia (CIN) or cervical cancer and a control group. All women resided in the same catchment area (Bronx Borough, New York City) and were of similar inner-city socioeconomic backgrounds representing a fairly homogenous population group. A cross-sectional sample of 235 women was recruited with informed consent. Plasma nutrient levels were measured by reverse-phase high pressure liquid chromatography under study codes. The mean plasma levels of carotenoids (beta-carotene, lycopene, and canthaxanthin), as well as alpha-tocopherol, were significantly lower in women with CIN and cervical cancer. In contrast, the mean plasma level of tau-tocopherol was higher among patients with CIN, while the mean plasma level of retinol was comparable among the groups. There were significant linear trends for all three carotenoids and quadratic trends for alpha- and tau-tocopherol with the degree of cervical histopathology. Plasma beta-carotene concentrations in cigarette smokers were significantly lower regardless of cervical pathology, whereas plasma lycopene and canthaxanthin levels were significantly lower in smokers with CIN. The findings of a decrease in all plasma antioxidant nutrient levels except tau-tocopherol in women with CIN and cancer suggest a potential role for antioxidant deficiency in the pathogenesis of CIN and carcinoma of the cervix, which requires further investigation.

Induction of transforming growth factor beta-1 in cervical intraepithelial neoplasia in vivo after treatment with beta-carotene.

Transforming growth factor (TGF) beta1 is a potent growth inhibitor of epithelial cells. Loss of responsiveness to TGF-beta1 and/or loss of TGF-beta1 itself may be important in the progression of cervical intraepithelial neoplasia to invasive cervical cancer. Retinoids have antiproliferative effects on epithelial cells and have been used as chemopreventive and chemotherapeutic agents for several human cancers. There is evidence that retinoids exert their effects by promoting the induction of TGF-beta. The aim of this study was to determine whether the expression of TGF-beta1 was altered in patients enrolled in a clinical trial designed to test the therapeutic efficacy of beta-carotene, a carotenoid metabolized to retinol, in cervical intraepithelial neoplasia. Using an immunohistochemical technique, tissues were stained with two types of antisera that react with the intracellular and extracellular forms of TGF-beta1. Matched cervical biopsies taken from 10 patients before and after treatment with beta-carotene were immunostained simultaneously to allow direct comparison of relative staining intensity. A significant increase in intracellular TGF-beta1 immunoreactivity was noted in cervical epithelial cells in patients with cervical intraepithelial neoplasia after treatment with beta-carotene (P = 0.003). These results demonstrate regulation of a TGF-beta isoform in vivo in humans in response to beta-carotene administered as a chemopreventive agent.

Sex difference in cellular retinol- and retinoic acid-binding proteins in human colon adenocarcinomas.

Human colon adenocarcinomas and adjacent non-cancerous, normal colon from the same patient were assayed for the presence and amounts of cellular binding proteins for retinol (CRBP) and retinoic acid (CRABP) by sucrose gradient analysis. In male patients, the mean concentrations of both CRBP and CRABP in the colon cancers were statistically significantly higher than in the adjacent normal colon. By contrast, in female colon cancers, the mean levels for both binding proteins were reduced approximately 2-fold, compared to the concentrations in the adjacent normal colon. These findings reveal an unexpected sex difference in the binding proteins for retinol and retinoic acid in human colon malignancies.

Human papillomaviruses of different types in precancerous lesions of the uterine cervix: histologic, immunocytochemical and ultrastructural studies.

In a prospective study of 34 women with abnormal Papanicolaou smears, biopsy and cervicovaginal lavage specimens were analyzed for the presence of human papillomaviruses (HPVs) by Southern blot analysis and probes for HPVs 6, 11, 16, and 18. In 22 of the 23 patients with cervical lesions (96%), HPV DNA was identified in one or more specimens. All patients in whom HPV DNA was found had either koilocytotic or dysplastic lesions on biopsy or Papanicolaou smear. Immunocytochemical demonstration of HPV in biopsy samples was associated with the presence of large amounts of HPV DNA and with the ultrastructural identification of viral particles. The presence of HPV DNA in cervical biopsy specimens was limited to discrete geographic areas of the cervix with histologic abnormalities. Although HPV 16 and other related HPV types were found in all cases of severe cervical intraepithelial neoplasia, the type of HPV present in a given specimen could not be predicted on the basis of morphologic, immunocytochemical, or electron microscopic findings. It is concluded that virtually all dysplastic lesions of the cervix contain HPV DNA, that HPV is thus likely to be a major etiologic agent in the pathogenesis of cervical dysplasia, and that histopathologic features are not predictive of HPV type.

Stringent criteria for histological diagnosis of koilocytosis fail to eliminate overdiagnosis of human papillomavirus infection and cervical intraepithelial neoplasia grade 1.

Overdiagnosis of HPV infection in cervical biopsies results in increased health care costs and unnecessary surgical procedures. Stringent criteria for histological diagnosis of koilocytosis were evaluated, using molecular detection of HPV DNA (polymerase chain reaction and Southern blot hybridization) as gold standard. Colposcopic biopsy specimens from 511 patients were studied, including 76 with referral diagnoses of negative cervix and 241 with CIN 1 or koilocytosis. Referral diagnoses for low-grade lesions failed to distinguish between HPV-infected and uninfected patients. False-positive rate for prediction of HPV infection was 74.8%. Biopsy specimens reevaluated using stringent diagnostic criteria showed increasing prevalence of HPV infection among patients whose biopsy specimens showed negative (43.7%), minimal (52.4%), or definite (69.5%) features of koilocytosis (P = .001). Similarly, subjects infected with high viral load or oncogenic HPV infection were more likely to be identified (P = .004 and .04, respectively). Despite increased predictive value of stringent diagnostic criteria, significant number of patients diagnosed as having CIN 1/koilocytosis (34.0%) did not in fact have HPV infection. Because most low-grade lesions spontaneously regress, patients with histological diagnosis of CIN 1 or HPV infection should be observed for a period of several months before definitive ablative treatment is undertaken.

Biologic characteristics of specific human papillomavirus types predicted from morphology of cervical lesions.

Human papillomavirus (HPV) DNA was detected by Southern blot hybridization in cervicovaginal lavage samples from 199 of 329 (60.5%) women attending a municipal hospital colposcopy clinic. Human papillomavirus was identified in 195 of 264 (73.9%) patients with a squamous intraepithelial lesion or cancer on biopsy or Papanicolaou smear (Bethesda system) compared with 11 of 65 (16.9%) without squamous intraepithelial lesion (P < .0001). The most common HPV type identified was HPV 16 (20.6% of positive samples), and 36.7% of isolates contained uncharacterized HPVs. Of women with cervical intraepithelial neoplasia (CIN) grade III or cancer, 23.4% were infected with HPV 16 compared with less than 4% with any other single HPV type. Based on biopsy diagnosis in patients infected with specific HPV types, HPVs 6 and 11 had low oncogenic potential; HPVs 18, 31, 35, and 45 had intermediate oncogenic potential; and HPVs 16 and 33 had high oncogenic potential. Hyperchromatic, unusually enlarged nuclei ("meganuclei"), and/or abnormal mitoses were found significantly more often in lesions infected with HPVs 16, 33, and 35 than in those infected with HPVs 6, 11, 18, 31, and 45, even in low-grade lesions, and may represent a histologic marker for HPVs with significant oncogenic potential. Human papillomavirus capsid protein was detected significantly less often by immunocytochemical staining in CIN I and CIN II lesions infected with HPVs 16 and 33 (8.3%) than in those infected with HPVs 6, 11, 18, and 31 (60%; P = .007), suggesting early abnormalities in cellular differentiation in lesions infected with highly oncogenic HPVs.

Malignant melanoma of the cervix uteri.

Primary malignant melanoma of the uterine cervix in a 70-year-old Negro female is reported. Only 9 other cases have been reported in the literature. The diagnosis, prognosis, and modern trends in therapy for this lesion are discussed.

Clinical usefulness of computerized colposcopy: image analysis and conservative management of mild dysplasia.

OBJECTIVES: The purposes of this study were to evaluate the clinical usefulness of computerized colposcopy and image analysis, to investigate the correlation between lesion size and grade of dysplasia, and to examine and record the colposcopic changes associated with progression or regression of cervical dysplasia. METHODS: Sixty-eight patients with a fully visualized squamocolumnar junction and a histopathologic diagnosis of mild dysplasia were serially monitored for a period of 1 year using computerized colposcopy and image analysis. All patients had baseline computer-assisted measurements of their cervical lesions and repeat measurements at 3-month intervals. RESULTS: The mean age of the patients was 31 years, and the mean size of the colposcopically visualized lesions was 58 mm2. During the 12-month observation, 5.9% of the lesions increased in size, 32.4% decreased in size, 13.2% remained unchanged, 20.6% disappeared, and 27.9% completely changed location. In patients with an increase in lesion size (N = 4), a repeat biopsy was performed, revealing a progression to moderate dysplasia. Treatment was withheld from patients whose lesions disappeared (N = 14), decreased in size (N = 22), or remained unchanged (N = 9). Active therapy was unnecessary in 66% of cases and repeat biopsy was avoided in 94.1%. CONCLUSIONS: Computerized colposcopy provides objective information and may be an adjunct to cytology and histopathology in some cases. Computerized colposcopy replaces subjective colposcopic evaluation with objective computer assessment and may hold promise for conservative management of cervical dysplasia.

Unruptured interstitial pregnancy. Diagnosis and treatment.

Symptoms and signs of interstitial pregnancy cannot be differentiated from symptoms and signs of other ectopic pregnancies. Unruptured interstitial gestations are usually diagnosed incidental to the work-up of a suspected ectopic gestation. Unlike some teaching, a characteristic pelvic mass was palpable in 3 of 4 cases. Incomplete induced abortion helped in diagnosing 2 cases. Ultrasound and laparoscopy will aid in establishing the correct diagnosis.

Placental and serum levels of carotenoids in preeclampsia.

OBJECTIVE: We compared placental tissue, maternal serum, and umbilical cord venous blood levels of four dietary carotenoids (alpha-carotene, beta-carotene, lycopene, and canthaxanthin) in normal pregnant women and those with preeclampsia. METHODS: Levels of alpha-carotene, beta-carotene, lycopene, and canthaxanthin were measured in placental tissue, maternal serum, and umbilical cord venous blood from 22 normal pregnant women and 19 women with preeclampsia. The criteria for recruitment included gestational age of 30-42 weeks, singleton pregnancy, intact membranes, absence of labor contractions, and absence of any other medical complication concurrent with preeclampsia. Carotenoids were measured using high-pressure liquid chromatography. RESULTS: All four carotenoids were detectable in human placental tissue, maternal serum, and umbilical cord venous blood samples. The levels of beta-carotene, lycopene, and canthaxanthin in placentas from preeclamptic women were significantly lower (P =.032, .009, and .013, respectively, by Mann-Whitney test) than those from normal pregnant women. Maternal serum levels of beta-carotene and lycopene were significantly lower (P =.004 and .008, respectively, by Mann-Whitney test) in women with preeclampsia. However, umbilical cord venous blood levels of these carotenoids were not significantly different between the two groups. CONCLUSION: Lower placental tissue and maternal serum carotenoid levels in women with preeclampsia suggest that oxidative stress or a dietary antioxidant influence might have an effect on the pathophysiology of preeclampsia.

Plasma concentrations of coenzyme Q10 and tocopherols in cervical intraepithelial neoplasia and cervical cancer.

Cervical intraepithelial neoplasia (CIN) may, at times, unpredictably progress to invasive carcinoma of the cervix. Epidemiological nutritional studies suggest that higher dietary consumption and circulating levels of certain micronutrients may be protective against cervical cancer. However, a preventive role of dietary antioxidants in CIN is not well established. The purpose of this cross-sectional study was to investigate the comparative plasma concentrations of three potent antioxidants, coenzyme Q(10,) alpha-tocopherol and gamma-tocopherol, in women with normal Pap smears and patients with a biopsy-confirmed histopathological lesion diagnosed as CIN or cervical cancer. Plasma concentrations of coenzyme Q(10,) alpha-tocopherol and gamma-tocopherol were measured by high-pressure liquid chromatography in both normal women without any history of abnormal Pap smears (n=48), and patients with histopathologically confirmed diagnoses of: (a) CIN I, n=98; (b) CIN II, n=49; (c) CIN III, n=10; and (d) cervical cancer, n=25. The mean plasma levels of coenzyme Q(10), alpha-tocopherol and gamma-tocopherol were significantly lower (P<0.001,<0.001, and<0.001, respectively by Kruskal-Wallis test) in patients with various grades of CIN and cervical cancer compared with controls. After controlling for age and smoking, an inverse association between histological grades of epithelial lesions and both plasma coenzyme Q(10) and alpha-tocopherol concentrations was observed. The low plasma concentrations of coenzyme Q(10) may be due to deficient dietary intake or a decrease in endogenous coenzyme Q(10) biosynthesis that may reflect increased utilization as a result of free radical reactive oxygen species induced oxidative stress. Further molecular studies on the mechanistic role of antioxidants in women with precancer cervical lesions are needed.

Plasma reduced and total ascorbic acid in healthy women: effects of smoking and oral contraception.

This is the first report of both reduced (RAA) and total (TAA) plasma ascorbic acid levels in women who smoke and are on oral contraception. 155 normal healthy subjects were investigated. A study questionnaire was completed including age, method of contraception, smoking status, and food intake at breakfast, prior to attending the clinic. Biochemical assays were promptly carried out without knowledge of subject's clinical or dietary status. One-way analysis of variance revealed that oral contraception per se, barrier or IUD methods have no effect on plasma ascorbic acid levels. The mean values (mg/dl +/- SD) for plasma RAA between smokers and nonsmokers were 0.512 +/- 0.241 and 0.601 +/- 0.263; and that for TAA were 0.565 +/- 0.232 and 0.682 +/- 0.231, respectively. Significant decreases in both plasma RAA (p less than 0.05) and TAA levels (p less than 0.001) were observed in smokers. Age was an interacting variable. No association of smoking or oral contraceptive use was seen with RAA or TAA levels among women less than 26 years, but decreases in both RAA and TAA levels were evident among smokers aged 26 years or older. The present study emphasizes the total exposure to smoking, e.g., pack years, is a significant confounding variable in the study of plasma ascorbic acid levels.

Pelvic angiography and the diagnosis of carcinoma of the uterine cervix.

The hospital records and pelvic arteriograms of 31 patients with suspected, persistent or recurrent carcinoma of the uterine cervix were reviewed. The method used was found to be highly accurate in diagnosing and localizing tumors and was considered most helpful in suspected pelvic wall recurrences. Tumor encasement of the vessels can be mimicked by perivascular, postoperative and post radiation fibrosis. Tumor vessels and tumor stain are the better angiographic indicators of the presence of recurrent or persistent disease.