LRIG1 and squamous epithelial uterine cervical cancer: correlation to prognosis, other tumor markers, sex steroid hormones, and smoking.

The aim is to evaluate LRIG1 as a prognosis predictor and correlations to cofactors in squamous cell cervical cancer. LRIG1 expression was studied in 128 cervical carcinomas and was compared with expression of nine other tumor markers. Smoking history was registered and pretreatment serum estradiol and progesterone levels were evaluated in 79 women. At clinical stage IB, 58% of the tumors showed LRIG1 expression, but there was a decline by increasing stage (33% in stage IV). Ninety percent of women with stage IB cancer and LRIG1 positivity survived, as compared to 64% without expression (P = 0.02). LRIG1 expression did not predict prognosis in advanced stages, but in stage IIA there was a marked relative difference, with 75% survival in tumors expressing LRIG1, as compared to 43% in those without. No correlation was found between LRIG1 and the other nine tumor markers studied. A high serum progesterone and smoking correlated to absent LRIG1 expression. We conclude that LRIG1 appears to be a significant prognosis predictor in early-stage cervical cancer, independent of the other tumor markers that were studied. Diminished expression in advanced stages and the inverse correlation to serum progesterone and smoking indicates that LRIG1 is a tumor suppressor in cervix.

Steroid production in different parts of malignant and benign ovarian tumors in vitro.

Pieces of ovaries and tumors from 45 patients (19 with malignant epithelial tumors, 14 with benign epithelial tumors, and 12 with normal postmenopausal ovaries) were incubated, and the release of steroid hormones from different parts of the tumors and from the contralateral ovaries was measured. Tumor tissue (mainly tumor cells with a small number of stromal cells), tumor base tissue (more stromal cells than tumor cells), and control ovaries were preincubated in oxygenated 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-minimum essential medium buffer at 37 degrees C for 30 min followed by a 3-h incubation in fresh, oxygenated medium. Progesterone, androstenedione, testosterone, and estradiol were measured in the medium by radioimmunoassay at the end of the incubation period. Malignant tumors released more progesterone and androstenedione than benign tumors or postmenopausal control ovaries. In contrast, benign tumors released more testosterone than malignant tumors or control ovaries. Release of estradiol was low and not significantly different among control ovaries and malignant and benign tumor tissue. Different parts of the tumors differed in steroid hormone release. Tissue samples containing more tumor cells than stromal cells released more progesterone than those with predominantly stromal cells. Thus, malignant tumors had an active steroid secretion. Progesterone was the main steroid released.

A population-based seroepidemiological study of cervical cancer.

The epidemiology of cervical cancer indicates the presence of a sexually transmitted risk factor, attributable at least in part to infection with human papillomavirus (HPV) type 16 or 18. We performed a seroepidemiological study of HPV and cervical cancer in the counties of Västerbotten and Norrbotten in Northern Sweden, a low-risk area for cervical cancer. Sera from 94 cases of incident cervical cancer were matched against 188 age- and sex-matched controls derived from a population-based blood bank. IgG and IgA antibodies were measured against a panel of 12 antigens derived from HPV types 6, 11, 16, or 18, as well as against Herpes simplex virus type 1 and 2, Chlamydia trachomatis, cytomegalovirus, Epstein-Barr virus, and bovine papillomavirus. Significantly increased relative risks (RRs) were found for IgG to HPV 16- or 18-derived antigens from the L1 (RR = 3.1), E2 (RRs = 2.8 and 9.2), and E7 (RRs = 3.8 and 2.7) open reading frames and for IgA to HPV 16-derived antigens from the E2 (RR = 3.3) and E6 (RR = 2.7) open reading frames. The highest RR (9.2, confidence intervals 4.4-19.4) was associated with IgG to an HPV 18 E2 antigen. Antibodies against cytomegalovirus, Herpes simplex virus type 2, Epstein-Barr virus, or bovine papillomavirus were, on their own, not significantly associated with cervical cancer, but seropositivity against multiple infections was associated with a successively increased relative risk. An increased risk was also found for IgG to Chlamydia trachomatis (RR = 1.7, confidence interval = 1.0-2.7). The results indicate that several HPV antibodies are strongly associated with cervical cancer, providing further seroepidemiological support for an etiological role of HPV in cervical cancer.

Steroid-hormone production and receptors in relation to ki-67, p53, s-phase fraction and ploidy in epithelial ovarian-tumors.

In this investigation, the in vitro production of progesterone and estradiol in ovarian tissues was studied for the first time in relation to the immunohistochemical expression of steroid hormone receptors, Ki-67, p53, DNA ploidy and S-phase fraction. Ovarian tissue from 44 women was examined. Steroid receptors were found more frequently in normal than in tumor ovaries. A substantial focal staining heterogeneity was demonstrated. Mucinous tumors were always progesterone receptor negative. Furthermore, the Ki-67 index was negatively correlated to the progesterone production of the tumor ovaries. Among the malignant tumors, all the high producers of progesterone expressing PR were low proliferating, diploid and p53-negative.

Release of epidermal growth-factor transforming growth-factor-alpha by ovarian-tumors in-vitro.

EGF-like activity was measured in media from 3-hour incubations of ovarian tissue from 27 patients with normal postmenopausal ovaries, malignant or benign epithelial tumours. EGF-like activity in the medium was measured using a radioreceptor assay. Malignant tumour tissue released significantly more EGF/TGF-alpha than benign tissues and aneuploid carcinomas more than diploid carcinomas. In spite of varying amounts of tumour cells there was a strong correlation in EGF/TCF-alpha release from the different tissue samples of each patient suggesting paracrine rather than autocrine regulation. The level of EGF-like activity may be a feature of the patient rather than of the tumour cells.

Blood group isoantigen expression during tumour progression of cervical neoplasia.

Twenty-nine cases of microinvasive squamous and 38 cases of progression from preinvasive to invasive carcinoma of the cervix were immunostained with monoclonal antibodies against blood group isoantigens for expression in normal, dysplastic and neoplastic epithelium. The results show a varying staining pattern without correlation to tumour progression, recurrence or survival.

Progesterone and estradiol stimulate release of epidermal growth factor/transforming growth factor alpha by ovarian tumours in vitro.

Previous studies have demonstrated locally increased steroid production and release of EGF/TGF-alpha in ovarian carcinomas. The influence of added steroid hormones on EGF-like activity in ovarian tumours in vitro was investigated. Using radioreceptor assay, EGF-like activity was measured in media from incubations of postmenopausal ovaries, benign and malignant epithelial ovarian tumours from 27 patients. The 3-hour incubations were performed with and without addition of progesterone, testosterone or estradiol. Addition of progesterone and estradiol significantly increased the release of EGF/TGF-a from benign tumour tissue but did not stimulate release from control ovaries. A stimulating effect was also noted for malignant tumour tissue but the increase was not significant. Addition of EGF or IGF-1 did not affect the release of steroid hormones from ovarian tumours. A positive correlation between progesterone and EGF-like activity was found in incubation media of peritoneal fluid cells from cancer patients.

Polyclonal cervical tumors detected by flow cytometry.

In a prospective study, the tumors of 167 patients with invasive squamous cell carcinomas of the uterine cervix were investigated by flow cytometric measurements of the DNA-content. In 37 patients (22%) 2 or more stemlines could be detected, indicating clonal heterogeneity. The proportion of multiple stemlines was equal in stages IB to III but significantly higher in stage IV (p less than 0.01). In 14 of the 37 polyclonal tumors the DNA-value of the second peak was twice the value of the first peak, suggesting possible polyploidization. For those tumors with evidence of polyploidization that had values in the diploid, tetraploid or octaploid region (n = 9) the mean age was 35.6 +/- 11.7 years and for grossly aneuploid tumors (n = 5) the mean age was 73 +/- 12.6 years. The differences of mean age between the two groups differed significantly (p less than 0.001). Flow cytometry proved to be a useful, rapid, and sensitive method for detection of multiple stemlines.

Blood group antigens in relation to DNA content, S-phase rate and heterogeneity, and their prognostic values in cervical carcinoma.

Evaluations were made of the expression of blood group antigens, DNA content and S-phase rate in a prospective study of 97 patients with squamous cell carcinoma of the uterine cervix. No correlation was found between loss of ABH expression and ploidy level or the degree of proliferation, whereas correlations were found to age and stage. In 11 (42%) of 26 tumors with two or three detectable stemlines, distinct areas with disparate ABH staining patterns were found, indicating genotypic and phenotypic heterogeneity within these tumors. In tumors with one stemline significantly fewer such tumors were found (p less than 0.01). Twenty-four early recurrences (within 20-36 months) after treatment were found. High S-phase rates (greater than 20%) were associated with a significantly increased recurrence rate (p less than 0.05). Loss of ABH antigen expression or ploidy level were not statistically correlated to early recurrences.

Oestrogens and clinical characteristics in endometrial carcinoma.

Serum concentrations of endogenously produced oestrogens were studied in 128 postmenopausal women with endometrial carcinoma. Radioimmunoassays of serum oestrone (E1) and oestradiol (E2) were performed on admission to hospital. Results showed a wide variation in serum concentrations of E1 and E2 in patients with endometrial carcinoma. Some patients had high E1 values and low E2, while none of the patients had low E1 and high E2 values. Hormonal levels were correlated with increasing BMI (E1; p less than 0.000001; E2; p less than 0.000001). Uterine cavity depth was also related to both E1 and E2 (E1; p less than 0.00001; E2; p less than 0.0003). Concentrations of E1 and E2 were higher in diabetic women than in the non-diabetic. (E1; p less than 0.002; E2; p less than 0.01). E2 concentration was higher in hypertensive patients than in the non-hypertensive patients (p less than 0.04). Parameters such as age, menstrual history, clinical stage and histopathology showed no significant correlation with hormone concentrations.

The reproducibility of flow cytometric analyses in human tumors. Methodological aspects.

Various methodological aspects of flow cytometry were studied in material from endometrial carcinoma, ovarian tumors and bladder carcinoma. Measurements of identical samples on two different occasions gave an excellent correlation of the obtained DNA values, r = 0.997 and a good reproducibility of the S-phase rate, r = 0.87. In large tumors different DNA values were found in 5/36 cases when central and surface biopsies were compared (indicating tumor heterogeneity) which stresses the importance of multiple biopsies. The S-phase rate of the surface biopsies was generally higher. Comparing staining with ethidium bromide and DAPI, a good correspondence of ploidy determinations in human bladder tumors was found, provided that diploid cells from the normal tissue component were used as internal reference. When ethidium bromide staining was used, there was a good agreement between the values of tumor ploidy obtained by external standardization using lymphocytes and internal standardization using diploid tumor cells, respectively. In DAPI staining with lymphocytes as an external standard the tumor ploidy was systematically overestimated by about 10% due to suboptimal staining of lymphocytes after 3 hours. This difference decreased after 18 hours of staining. Determination of S-phase fraction showed a good correlation between DAPI and ethidium bromide stained bladder tumor samples (r = 0.86). Human lymphocytes as an external standard showed good reproducibility. In conclusion, flow cytometric measurements of the ploidy level and S-phase rate are highly reproducible provided that tumor heterogeneity is taken into account and proper preparation and standardization methods are used.

Progesterone androstenedione testosterone and clinical characteristics in endometrial carcinoma.

Serum concentrations of progesterone (P), androstenedione (A) and testosterone (T) were measured in 128 postmenopausal women with endometrial adenocarcinoma. The correlations between these hormones were studied and related to such clinical parameters as: stage, histopathology, age, gynaecological history, body mass index and related diseases. In previous studies we have shown the relations between oestron (E1) and oestradiol (E2) serum concentration and clinical parameters. A clear correlation was seen between E1 and E2. Both E1 and E2 also strongly correlated with BMI. In this study androstenedione is found to correlate with E1 and E2, p less than 0.03 and p less than 0.02 resp. Progesterone and E1 were correlated, p less than 0.01, but not P and E2. Testosterone correlated with E1, p less than 0.002, E2, p less than 0.003 and A, p less than 0.02. None of the hormones P, A or T correlate with BMI. Androstenedione and age, and A and menopausal years showed a negative correlation, p less than 0.01 for both. Testosterone correlated with menopausal years, p less than 0.03; and correlated negatively with years of menstruation, p less than 0.03. These results do not support the hypothesis that A is the exclusive precursor for conversion to E1 in fat tissue. The correlation between P and E1 could indicate a relation to steroids higher up in the synthesis chain. The influence of P, A and T on the investigated clinical factors can not be strong, since correlations are virtually missing.

S-phase rate as a predictor of early recurrences in carcinoma of the uterine cervix.

Flow cytometric measurements of ploidy level and S-phase rate were performed in a prospective study of 165 patients with cervical squamous cell carcinoma. Patients were followed-up for 24 months after initial treatment. There was no significant difference in recurrence rate between patients with peri-diploid tumors (1.8c-2.2c) and aneuploid tumors. The S-phase rate (evaluable in 133 cases), which previously has been studied only occasionally in cervical carcinomas, was correlated with early relapses. Significantly more relapses were found in tumors with S-phase rates greater than or equal to 20% than among tumors with S-phase rates less than 20% (chi 2 = 9.54, p less than 0.01). A higher relapse rate among tumors with S-phase rates greater than or equal to 20% was found even after taking staging into consideration (Cochran-Mantel-Haenszel test, p less than 0.01). Thus, besides staging, evaluation of the S-phase rate yielded additional prognostic information.

Blood group isoantigen and involucrin expression in cervical intraepithelial neoplasia.

258 patients with dysplastic cervical lesions reclassified as CIN 1-3 and AIM 1-3 and with up to ten years of follow-up were immunostained with monoclonal antibodies against blood group isoantigens and involucrin for expression in normal and dysplastic squamous epithelium. The results show a varied staining pattern with a majority of unchanged staining properties in normal and dysplastic epithelium, the same frequency of increase as decrease in dysplasia and no correlation to progression or recurrence, in variance with previous reports.

Peritoneal fluid and HCG influence on steroid production from ovarian tumor tissue, in vitro.

Steroids are produced by malignant and benign epithelial ovarian tumor tissue in vitro, but the regulation is unknown. The effect of peritoneal fluid and beta-HCG on steroid production was analysed. Tissue from 17 malignant or benign epithelial ovarian tumors and 6 normal postmenopausal ovaries were incubated. In 11 cases tissue was incubated with and without addition of the patient's own peritoneal fluid (Part I). Tissue from 22 ovaries was incubated with and without HCG (100 IU/ml medium), (Part II). Furthermore, the release from tumorous and control ovaries of beta-HCG, progesterone, androstenedione and estradiol measured using radioimmunoassay (Part III). Peritoneal fluid stimulated the release of progesterone from both malignant and benign tissue and androstenedione or estradiol stimulated release from benign tissue (Part I). There was no stimulatory effect of HCG on steroid release from malignant tissue. HCG stimulated release of estradiol in benign tissue (Part II). All malignant tumors and 4 out of 6 benign tumors released beta-HCG (Part III). There seems to exist factors in peritoneal fluid which are able to stimulate steroid production from ovarian tumors.

Age, menopausal status and DNA-content in endometrial adenocarcinoma.

In a prospective study of 283 women with endometrial carcinoma, 29 of whom were premenopausal and 254 postmenopausal, the DNA content and S-phase rate were measured by flow cytometry. Over 90% of the tumors in premenopausal women were peri-diploid and the remaining tumors were tetraploid, whereas 23% of the tumors in post-menopausal women were grossly aneuploid (p less than 0.01). Similar differences were found between women younger or older than 50 years. The results indicate that the hormonal status and/or age are of importance for the development of genetically different forms of endometrial carcinomas, as judged from the DNA-distribution.

EGF/TGF-alpha and progesterone in urine of ovarian cancer patients.

EGF/TGF-alpha and progesterone were measured in the urine of 74 ovarian carcinoma patients, 21 postmenopausal women with non-gynecological disseminated cancer, 20 premenopausal and 20 postmenopausal controls. Radically operated women excreted significantly less EGF/TGF-alpha into urine than women with residual tumour mass. The patients who died from ovarian carcinoma had significantly higher concentrations of growth factor in urine than patients who were alive and disease-free at follow-up. The highest urinary concentrations were found in the premenopausal control group and among women with non-ovarian malignancies. A significant correlation between concentrations of progesterone and EGF/TGF-alpha in urine was noted.

Influence of oestrogen on growth of endometrial carcinoma.

The influence of endogenously produced oestrogen on the growth of endometrial carcinoma was studied in postmenopausal 128 patients with endometrial adenocarcinoma. Plasma concentration of oestrone (E1) and oestradiol (E2) showed wide variations. Hormone levels were analysed in relation to growth rate, expressed as S-phase rate measured by flow cytometry, and to ploidy level. When the whole unclassified group was studied, no statistical relationship between E1 and E2 levels and S-phase rates were found. However, when peridiploid tumors (1.8-2.2 c) were divided according to histopathological grades, well differentiated tumors with low oestradiol concentrations (less than 60 pmol/I) had significantly lower S-phase rates than those with higher oestradiol levels (p less than 0.01). Aneuploid tumors showed high S-phase rates regardless of plasma oestradiol concentrations.

Prognostic indices in endometrial adenocarcinoma stages I and II. A study based on clinical, histopathological and flow cytometric variables.

In a prospective study including 447 patients with endometrial adenocarcinoma stages I and II, clinical and flow cytometric variables were studied in univariate and multivariate analyses. During the follow-up period of 3 to 65 months, 41 patients died of their disease and 26 patients died of intercurrent disease. Based on the results of multivariate analyses, five different prognostic indices were constructed. Using these indices, individual index values were calculated for each patient. The index values were correlated with survival and were used to identify a small high-risk group (10% of the patients) and a large low-risk group (90% of the patients). Indices 1 and 2 were constructed for pre-treatment situations. Index 1 included age and histopathologic grade and identified a small high-risk group and a large low-risk group with a 3-year survival of 62% and 91% respectively. Index 2 also comprised S-phase fraction and distinguished a high-risk group with a 3-year survival of 54% from a low-risk group with a 3-year survival of 93%. Indices 3 and 4 were constructed for patients after surgery, when the degree of residual myometrial tumor invasion was known. Index 3 included age, histopathologic grade and residual myometrial invasion rendering a 3-year survival of 44% and 93% for the high- and low-risk groups respectively. Index 4 also comprised S-phase fraction and identified a high-risk group and a low-risk group with 3-year survival figures of 59% and 96%, respectively. Index 5 included age and histopathologic grade and was constructed for post-treatment situations in unoperated patients. The index separated a high-risk group from a low-risk group with a 3-year survival of 56% and 88%, respectively. In summary, the results from our study show that the prognostic indices made it possible to identify high and low-risk groups in different clinical situations which may prove useful in selecting candidates for future trials with adjuvant or reduced treatment.

Double HPV infection in African cervical cancer detected by polymerase chain reaction.

Biopsy samples from 13 Kenyan patients with squamous cell carcinoma of the cervix were analysed for the presence of type specific HPV DNA by polymerase chain reaction (PCR). HPV 16 was confirmed in 11 (85%) and HPV 18 in 9 (69%) samples. HPV 6 DNA was detectable in only 3 (23%) samples and no HPV 33 was found. Infection with either HpV16 or 18 was seen in 12 (92%) and infection with both in 8 (62%) cases. The prevalence of double infection found is higher than in previous reports. The significance of this and possible effects of parity on cervical neoplastic changes are discussed.