Lucien Frappart

ACCURACY OF VISUAL SCREENING FOR CERVICAL NEOPLASIA: RESULTS FROM AN IARC MULTICENTRE STUDY IN INDIA AND AFRICA

Visual inspection‐based screening tests, such as visual inspection with 4% acetic acid (VIA) and with Lugols iodine (VILI), have been proposed as alternatives to cytology in mass screening programs. To date, there is only limited information on the accuracy of these tests in detecting High‐grade Squamous Intraepithelial Lesions (HSIL). Eleven cross‐sectional studies involving 56,939 women aged 25–65 years were conducted in Burkina Faso, Congo, Guinea, India, Mali and Niger to evaluate the accuracy of VIA and VILI performed by health workers. A common protocol and questionnaire was used. For final diagnosis, all women were investigated with colposcopy and biopsies were taken when necessary. Data from the studies were pooled to calculate sensitivity, specificity and predictive values of the tests for the detection of HSIL. Of the screened women, 16.1% and 16.4% were positive on examination using, respectively, VIA and VILI; 1,063 were diagnosed with HSIL. The pooled sensitivity, specificity, positive and negative predictive values for VIA were 76.8% (95% CI: 74.2–79.4%), 85.5% (95% CI: 85.2–85.8%), 9.4% (95% CI:8.8–10.8%) and 99.5% (95% CI:99.4–99.6%), respectively. The values were 91.7% (95% CI: 89.7–93.4%), 85.4% (95% CI: 85.1–85.7%), 10.9% (95% CI: 10.2–11.6%) and 99.8% (95% CI:99.7–99.9%), respectively for VILI. The range of sensitivity and specificity for VIA was 56.1–93.9% and 74.2–93.8%, respectively, between studies and were 76.0–97.0 % and 73.0–91.3% for VILI. VILI had a significantly higher sensitivity than VIA in detecting HSIL, but specificity was similar. VILI appears to be a more accurate visual test for use in screening and treatment programs in low‐resource settings.

Down-regulation of BRCA1 in human sporadic breast cancer; analysis of DNA methylation patterns of the putative promoter region

Germ-line alterations of BRCA1 are responsible for about 50% of familial breast cancers. Although its biological function(s) has not yet been fully determined, it has been suggested that it may act as a tumor suppressor gene in breast and ovarian cancers. In sporadic breast cancers alterations of BRCA1 have not been detected and in vitro experiments have indicated that BRCA1 negatively regulates cellular proliferation. The present study was designed to identify and quantify, the BRCA1 mRNA levels, in normal and neoplasic human breast tissue. BRCA1 mRNA molecules were quantified using competitive reverse transcriptase PCR assays. DNA methylation patterns of this gene have been analysed by Southern blot experiments using methylation sensitive restriction enzymes. We found that BRCA1 mRNA levels were significantly lower in sporadic breast cancers (37 cases analysed, 24 cases of invasive ductal carcinomas not otherwise specified (NOS), two lobular carcinomas in situ two medullary carcinomas, four invasive lobular carcinomas, two invasive mucinous carcinomas and three invasive ductal carcinomas with predominantly in situ component) compared with normal breast tissues (P=0.0003). This down-regulation of BRCA1 is observed in all histologic types analysed. In invasive ductal carcinomas NOS, this down-regulation does not correlate with any of the prognostic factors studied (tumor size, node status, histologic grade, hormone receptor status). In the samples analysed, alterations of DNA methylation patterns were not dectected in the vicinity of the major transcription start site. These data suggest the involvement of BRCA1 in the carcinogenesis of these histologic types.

Accuracy of human papillomavirus testing in primary screening of cervical neoplasia: Results from a multicenter study in India

The knowledge that cervical neoplasia are caused by human papillomavirus (HPV) infection has led to the evaluation of its role in screening. We evaluated the accuracy of HPV testing by Hybrid capture II (HC II) method in detecting cervical intraepithelial neoplasia grade 2 and 3 (CIN 2 and 3) lesions in 4 cross‐sectional studies with common protocol and questionnaire in 3 different locations (Kolkata, Mumbai and Trivandrum) in India. These studies involved 18,085 women aged 25–65 years. The reference standard for final diagnosis was a combination of colposcopy/biopsy. All women were investigated with colposcopy and 3,116 received directed biopsy. The sensitivity of HPV testing for detecting CIN 2–3 lesions varied from 45.7% to 80.9% across the study sites; the specificity varied from 91.7% to 94.6% and the positive predictive value from 6.7% to 13.7%. Retesting of 298 randomly chosen denatured samples in France revealed an agreement rate of 85.9% and a κ‐value of 0.72. Although HPV testing seems to be a promising approach for cervical cancer prevention, a large range in sensitivity was observed in our study, possibly due to variations in the quality of specimen collection and reference standards. A higher sensitivity was associated with the center performing the test well. Further developments in terms of more reproducible, less expensive and less sophisticated testing are essential to make the test feasible and effective in low‐resource settings.

Follicular viability and morphology of sheep ovaries after exposure to cryoprotectant and cryopreservation with different freezing protocols

OBJECTIVE To test the toxicity of cryoprotectant in sheep ovarian tissue and to determine optimal conditions for freezing hemiovary cortex. DESIGN Small follicles (<60 microm in diameter) were isolated enzymatically for viability testing. Dead and live follicles were identified by using trypan blue staining, and follicle morphology was examined histologically. SETTING Centre hospitalo-universitaire de Biologie de la Reproduction, Hôpital Edouard Herriot, Lyon, France. ANIMAL(S) Lambs 5 to 6 months of age. INTERVENTION(S) Two-millimeter slices of hemiovarian cortex were prepared for cryoprotectant toxicity tests and freezing procedures. MAIN OUTCOME MEASURE(S) Follicular mortality and histologic structure. RESULT(S) For freezing procedures, the concentration of cryoprotectant was increased to 2 M on the basis of results of cryoprotectant toxicity tests in fresh tissues. Follicular mortality rates were 4.6% with of 2 M dimethyl sulfoxide (DMSO) and 3.8% with 2 M of propylene glycol (PROH). After freezing with semiautomatic seeding, follicular mortality rates were 8.4% (2 M of DMSO) and 12.4% (2 M of PROH). Tissue morphology was well preserved with 1.5 M of DMSO or PROH. With 1.5 M DMSO, results of the slow cooling protocol (2 degrees C/min) without seeding and the standard very slow cooling protocol (0.3 degrees C/min) were similar. CONCLUSION(S) Optimal survival of primordial follicles in the sheep was obtained by using a slow cooling protocol with semiautomatic seeding at 2 M of DMSO.

Initial results from a randomized trial of cervical visual screening in rural south India

The impact of a single round of screening of visual inspection with acetic acid (VIA) on cervical cancer incidence and mortality was investigated in a cluster randomized trial in south India. Women 30–59 years of age in 113 clusters in Dindigul District were randomized to VIA screening (57 clusters, 48,225 women) by nurses and to a control group (56 clusters, 30,167 women). 30,577 eligible women were screened between May 2000 and April 2003; 2,939 (9.6%) screen‐positive women were investigated with colposcopy by nurses and 2,777 (9.1%) women had biopsy. CIN 1 was diagnosed in 1,778 women, CIN 2‐3 lesions were found in 222, and there were 69 screen detected invasive cervical cancers. The detection rates of lesions per 1,000 screened women were 58.2 for CIN 1, 7.3 for CIN 2‐3, and 2.3 for invasive cancer. The detection rate of high‐grade lesions in our study was 2–3‐fold higher than those observed in repeatedly screened populations in developed countries. 71% of women with CIN 1 and 80% of those with CIN 2‐3 lesions accepted cryotherapy provided by nurses and surgical treatment by mid‐level clinicians. Overall, 97 and 34 incident cervical cancer cases were observed in the intervention and control arms, respectively. The intervention arm accrued 124,144 person years and the control arm accrued 90,172 during the study period. The age standardized cervical cancer incidence rates were 92.4/100,000 person‐years in the intervention and 43.1/100,000 in the control arms. In the screened arm, 35.0% of cases were in Stage I as opposed to none in the control arm. The preliminary findings from our study indicate that not only is a VIA‐based screening programme feasible, safe and acceptable to a population in rural settings, it also results in early detection of cervical neoplasia.

Glucocorticoid therapy of antigen-induced arthritis depends on the dimerized glucocorticoid receptor in T cells

Despite several side effects, glucocorticoids (GCs) have been widely used for 60 y to treat rheumatoid arthritis on the basis of their antiinflammatory effects. However, the cells targeted by GCs and the transcriptional mechanisms underlying their actions through the glucocorticoid receptor (GR) in steroid therapy remain poorly defined. Using cell type-specific GR-deficient mice subjected to antigen-induced arthritis (AIA) as a model of human rheumatoid arthritis, we show that GC action on T cells but not myeloid cells is critical for therapeutic intervention in AIA. Furthermore, the resistance of mice expressing a DNA binding-defective GR (GRdim) to GC treatment reveals that dimerization of the GR is indispensable for the antiinflammatory effects. In these mice, the GC-induced suppression of TH1 and TH17 cell-derived proinflammatory cytokines is impaired. Our finding that IL-17A−/− mice are resistant to GC therapy, whereas IFN-γ−/− mice respond as efficiently as WT mice implies that IL-17–producing T cells and not IFN-γ–producing T cells are the most important targets for an efficient GC therapy. The present studys identification of the critical cell type and the mode of GR action in steroid therapy of AIA significantly advances our understanding of steroid therapy and should lead to therapies with greater efficiency and fewer side effects.

Morphological alterations and DNA fragmentation in oocytes from primordial and primary follicles after freezing-thawing of ovarian cortex in sheep

OBJECTIVE To evaluate DNA fragmentation in the oocyte of primordial and primary follicles and morphology of these follicles after freezing and thawing of ovarian cortex in sheep using two freezing protocols. DESIGN Fragmentation of DNA was evaluated by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) technique. SETTING Fertility clinic in a large university hospital. ANIMALS Five- to 6-month-old lambs. INTERVENTION(S) Two-millimeter-thick slices of hemi-ovary cortex were prepared. MAIN OUTCOME MEASURE(S) Histological structure and DNA fragmentation. RESULT(S) In the frozen fragments, the percentage of morphologically normal follicles was significantly lower for both protocols compared with the case of the control group of fresh fragments. There was no significant difference between the two types of freezing protocols (60.4% +/- 13.2% vs. 68.4% +/- 13.7%). However, the distribution of abnormalities (nucleus, cytoplasm, and nucleus and cytoplasm) was dissimilar. The results of the TUNEL technique for the three groups showed no significant difference, but the percentage of the TUNEL-positive follicles was slightly lower for the frozen fragments for both protocols with respect to the control group. CONCLUSION(S) The freezing and thawing process of the ovarian cortex does not induce fragmentation of the DNA on the oocyte of primary and primordial follicles.

Regional methylation of the 5′ end CpG island of BRCA1 is associated with reduced gene expression in human somatic cells

In mammalians, demethylation of specific promoter regions often correlates with gene activation; inversely, dense methylation of CpG islands leads to gene silencing, probably mediated by methyl‐CpG binding proteins. In cell lines and cancers, inhibition of tissue‐specific genes and tumor suppressor genes expression seems to be related to such hypermethylation. The 5’ end of the breast cancer predisposition gene BRCA1 is embedded in a large CpG island of ~2.7 kb in length. In human sporadic breast cancers, the down‐regulation of BRCA1 does not seem to be related to BRCA1 gene alterations. Southern blot analysis and the bisulfite sequencing method indicate that the BRCA1 CpG island is regionally methylated in all human tissues analyzed and unmethylated in the gametes, suggesting a role for DNA methylation in the control of gene expression. We have therefore investigated the potential role of methyl‐CpG binding proteins in the regulation of BRCA1 gene expression. In vitro, partial methylation of constructs containing this region strongly inhibits gene expression in the presence of MeCP2 protein. Moreover, in the five human cell lines analyzed, chemically induced hypomethylation is associated with BRCA1 gene activation. These data suggest that methyl‐CpG binding proteins might be associated with the control of BRCA1 gene expression and that methyl‐DNA binding proteins may participate in the regulation of gene expression in mammalian cells.–Magdinier, F., Billard, L.‐M., Wittmann, G., Frappart, L., Benchaïb, M., Lenoir, G. M., Guérin, J. F., Dante, R. Regional methylation of the 5’ end CpG island of BRCA1 is associated with reduced gene expression in human somatic cells. FASEB J. 14, 1585–1594 (2000)

Human breast tumors override the antiangiogenic effect of stromal thrombospondin-1 in vivo.

The antiangiogenic extracellular matrix protein thrombospondin‐1 (TSP‐1) inhibits tumor growth and metastasis in animals. However, the clinical relevance of such findings are equivocal as increased stromal TSP‐1 expression has been associated with either good or poor prognosis. In an effort to obtain a more integrated understanding of the role of TSP‐1 in breast cancer, we first used a breast tumorigenesis model in which tumor‐associated stromal fibroblasts were engineered to produce high levels of TSP‐1. We demonstrate here that stromal TSP‐1 delayed human MDA‐MB‐231/B02 breast tumor growth. However, this delay in MDA‐MB‐231/B02 tumor growth upon exposure to TSP‐1 was associated with an increased vascular endothelial growth factor (VEGF) expression in tumor cells themselves, leading to a tumor growth rate comparable to that of tumors whose fibroblasts did not overproduce TSP‐1. Clinical evidence also suggested that primary breast carcinomas have adapted to escape the effects of stromal TSP‐1. TSP‐1 was found to be expressed in the stroma of human breast carcinomas where, although its level correlated with decreased vascularization, it was unexpectedly associated with a reduction of relapse‐free survival. In metastatic axillary lymph nodes, tumor cells expressed high levels of VEGF and TSP‐1 expression were no longer associated with a decreased vascularization. Overall, these results suggest that a resistance may develop early in human breast cancers as a result of high in situ exposure to stromal TSP‐1, leading to disease progression.

Involvement of DNA methylation in the control of the expression of an estrogen‐induced breast‐cancer‐associated protein (pS2) in human breast cancers

pS2 gene has been used to investigate the relationship between alterations of DNA methylation patterns in human tumors and gene expression. The expression of pS2, which is transcriptionally controlled by estrogens in breast cancer cell lines, is restricted to estrogen‐receptor‐rich human breast tumors. We found that the CCGG site within the promoter/enhancer sequence of pS2 was hypomethylated in estrogen‐receptor‐rich breast tumors expressing this gene. The amount of DNA molecules unmethylated at this site was related to the amount of pS2 mRNA detected in the samples. The demethylation of this region, which contains the estrogen responsive element, was confirmed by genomic sequencing. Transient expression of functional human estrogen receptors stimulated the expression of the endogenous pS2 in HeLa cells, but failed, in BT‐20 cells, to stimulate expression of this gene. Since the promoter/enhancer region of pS2 is unmethylated in HeLa cells and methylated in BT‐20 cells, these data also support the hypothesis that DNA methylation might be involved in the control of pS2 expression. J. Cell. Biochem. 65:95–106.