Cervical trophoblasts for non-invasive single-cell genotyping and prenatal diagnosis.

OBJECTIVE: We aimed at developing a method to recover trophoblastic cells from the cervix through a completely non-invasive approach and obtaining a genetic proof of their fetal nature implying that they can be used for non-invasive prenatal diagnosis (NIPD). METHODS: We studied obstetrical samples from 21 pregnant women between 8 and 12 weeks of gestation scheduled for chorionic villus sampling or undergoing elective termination of pregnancy. A cytobrush was used to extract cells from the external parts of the cervix and transferred to 10 ml of preservative solution. Cells were layered on filters with 8 microns pores using the ISET system (Isolation by SizE of Tumor/Trophoblastic cells) and stained. Putative fetal cells were collected by single cell laser-assisted microdissection and identified as fetal or maternal cells by Short Tandem Repeat genotyping. NIPD was blindly performed on 6 mothers at risk of having a fetus with Cystic Fibrosis or Spinal Muscular Atrophy. RESULTS: Trophoblastic cells were recovered from all tested cervical samples with a frequency of 2-12 trophoblasts per 2 ml. NIPD was blindly obtained and verified in 6 mothers at risk of having a fetus with Cystic Fibrosis or Spinal Muscular Atrophy. DISCUSSION: Although larger confirmation studies are required, this is the first report providing a solid proof of principle that trophoblasts can be consistently and safely recovered from cervical samples. Since they are a source of pure fetal DNA, i.e. fetal DNA not mixed with maternal DNA, they constitute an ideal target to develop NIPD of recessive diseases, which is a technical challenge for methods based on cell free DNA.

[Expected impact of a quadrivalent HPV vaccine in France]. / Modélisation de l'impact de la vaccination HPV quadrivalente en France.

OBJECTIVE: To assess the expected impact in France of a quadrivalent HPV 6/11/16/18 vaccine on the occurrence of genital HPV-induced lesions in women. METHODS: A Markov model based on a quadrivalent vaccination of 14-year-old girls as recommended in France was performed to assess the number of subjects needed to vaccinate to prevent an HPV-related event during their lifetime and the expected annual number of cases which could be prevented by vaccination. This model was based on prevalence data reported in four large French studies (EDiTH I-IV) reporting an HPV 6/11/16/18 prevalence of 82% (95% CI: 78.5-85.1) in cervical cancer (CC), 64% (95% CI: 59.7-68.1) in CIN2/3, 34% (95% CI: 28.9-38.1) in low-grade squamous intraepithelial lesions (LSIL) and 83% (95% CI 77.6-87.8) in female external acuminata condylomata (EAC) cases. RESULTS: Using a theoretical vaccine efficacy of 100%, 130 young women need to be vaccinated to prevent a case of CC, 17 for a case of CIN2/3 and 13 for a case of EAC. Immunization of 80% of 14-year-old girls could prevent 2495 CC (72%), 17,985 CIN2/3 (54%), 8004 CIN1 (27%), and 22,531 EAC female cases (65%) in France annually. CONCLUSION: A good adhesion to the preferentially recommended HPV quadrivalent vaccination would thus substantially reduce the burden of female genital lesions in France.

[Interest of new biomarkers in cervical precancerous lesion management]. / Intérêt des nouveaux marqueurs dans la prise en charge des lésions précancéreuses du col utérin.

The difficulties of cytological identification of abnormal cells, especially in case of "minor atypias", have led to search for new markers able to make cytological screening more sensitive without decrease of specificity. To date, p16 seems to be the most interesting one. It is currently used in immunohistochemistry on cervical biopsies to distinguish immature metaplasias from high-grade lesions. In cytology, its interest is not yet well defined. According to recent papers, p16 overexpression could improve the efficacy of cytological diagnosis. Further studies are required to make positive recommendations.

Human papillomavirus testing improves the accuracy of colposcopy in detection of cervical intraepithelial neoplasia.

To assess the performance of human papillomavirus (HPV) testing and colposcopy in detection of cervical pathology. A series of 389 women referred for colposcopy due to an abnormal Pap smear had cervical swabs analyzed for oncogenic (high-risk [HR]) HPV types using Hybrid Capture II (HC2) assay. Loop electrical excision procedure cone biopsy (88%) or colposcopic biopsy (11%) was used as the gold standard. Of the atypical squamous cells of undetermined significance (ASCUS) smears, 48% were positive for HR HPV, as compared to 76.3% of low-grade squamous intraepithelial lesions (LSIL) smears. HR HPV was detected in 66.7% and 90% of patients with cervical intraepithelial neoplasia (CIN) 1 and CIN2 (or higher), respectively. The sensitivity of the Pap smear using an ASCUS threshold in detecting high-grade CIN was 94.5% (95% confidence intervals (CI): 91-97%) and that of colposcopy 98.5% (95% CI: 95-99%). The respective specificities were 30% (95% CI: 17-28%) and 35.6% (CI: 29-42%). HC2 test had comparable sensitivity, 90% (95% CI: 85-93%), but higher specificity, 54.3% (95% CI: 47-61%). Combining HC2 test with Pap increased specificity, 66.7% and 41.3% for ASCUS and LSIL cutoff, respectively. The minor-abnormality threshold together with HC2 increased specificity of colposcopy with no changes in sensitivity. High viral load (>100 relative light unit/positive control) was associated with significant disease. HPV DNA testing improves the accuracy of colposcopy in the detection of high-grade CIN in women with ASCUS or LSIL smears.

Liquid-based cytology for primary cervical cancer screening: a multi-centre study.

The aim of this six-centre, split-sample study was to compare ThinPrep fluid-based cytology to the conventional Papanicolaou smear. Six cytopathology laboratories and 35 gynaecologists participated. 5428 patients met the inclusion criteria (age > 18 years old, intact cervix, informed consent). Each cervical sample was used first to prepare a conventional Pap smear, then the sampling device was rinsed into a PreservCyt vial, and a ThinPrep slide was made. Screening of slide pairs was blinded (n = 5428). All non-negative concordant cases (n = 101), all non-concordant cases (n = 206), and a 5% random sample of concordant negative cases (n = 272) underwent review by one independent pathologist then by the panel of 6 investigators. Initial (blinded) screening results for ThinPrep and conventional smears were correlated. Initial diagnoses were correlated with consensus cytological diagnoses. Differences in disease detection were evaluated using McNemar's test. On initial screening, 29% more ASCUS cases and 39% more low-grade squamous intraepithelial lesions (LSIL) and more severe lesions (LSIL+) were detected on the ThinPrep slides than on the conventional smears (P = 0.001), including 50% more LSIL and 18% more high-grade SIL (HSIL). The ASCUS:SIL ratio was lower for the ThinPrep method (115:132 = 0.87:1) than for the conventional smear method (89:94 = 0.95:1). The same trend was observed for the ASCUS/AGUS:LSIL ratio. Independent and consensus review confirmed 145 LSIL+ diagnoses; of these, 18% more had been detected initially on the ThinPrep slides than on the conventional smears (P = 0.041). The ThinPrep Pap Test is more accurate than the conventional Pap test and has the potential to optimize the effectiveness of primary cervical cancer screening.

[Liquid phase cytology in the primary screening for cervical cancer: a multicenter study]. / Cytologie en phase liquide dans le cadre du dépistage primaire du cancer du col utérin: une étude multicentrique.

The aim of this six-centre, split-sample study was to compare ThinPrep fluid-based cytology to the conventional Papanicolaou smear. Six Cytopathology laboratories and 35 Gynaecologists participated. 5428 patients met the inclusion criteria. Each cervical sample was used first to prepare a conventional Pap smear, then the sampling device was rinsed into a PreservCyt vial, and a ThinPrep slide was made. Screening of slide pairs was blinded. On initial screening, 29% more ASCUS and 39% more low-grade squamous intraepithelial lesions (LSIL) and more severe lesions (LSIL+) were detected on the ThinPrep slides than on the conventional smears (p = 0.001). Independent and consensus review confirmed 145 LSIL + diagnoses; of these, 18% more had been detected initially on the ThinPrep slides than on the conventional smears (p = 0.041). The ThinPrep Pap Test is more accurate than the conventional Pap Test and has the potential to optimize the effectiveness of primary cervical cancer screening.

Heterogeneity of DNA distribution in diploid cells: a new predictive discriminant factor for solid tumour behaviour.

Spatial nuclear DNA heterogeneity distribution of Feulgen-stained DNA diploid cells was studied by image cytometry in voided urine of 119 patients without bladder tumour (n = 20) and with initial (n = 23) or previous (n = 76) diagnosed bladder tumour. For each patient, repetitive DNA measurements were performed during 1-4 years of follow up. Only cells of diploid DNA histograms and diploid subpopulations of aneuploid DNA histograms were used for analysis. DNA heterogeneity distribution of these diploid cells was quantified by statistical parameters of each nuclear optical density distribution. Discriminant analysis was performed on three groups of DNA histograms. Group A (n = 44): aneuploid DNA histograms of patients with bladder tumour. Group D (n = 55): 38 diploid DNA histograms of the 20 patients without bladder tumour (subgroup D1) and 17 diploid DNA histograms of patients with a non-recurrent bladder tumour (subgroup D2). Group R (n = 27): diploid DNA histograms of patients with bladder tumour recurrence. No statistically significant discriminant function was found to separate D1 and D2. However, the first canonical discriminant function C1 differentiated diploid cells of diploid DNA histograms (group D and group R) from diploid cell subpopulations of aneuploid DNA histograms (group A). Mean C1 values were 1.06, 0.84 and -1.45 for groups R, D and A, respectively. The second canonical discriminant function C2 differentiated diploid DNA histograms of patients with bladder tumour recurrence (group R) from diploid DNA histograms of patients without bladder tumour or without bladder tumour recurrence (group D). Mean C2 values were 1.78 and -0.76 for groups R and D, respectively. In 95% confidence limit, the rate of rediscrimination using the two first canonical discriminant functions C1 and C2 were 86.4, 74.5 and 74.1% for groups A, D and R, respectively. Percent of "grouped" cases correctly classified was 78.6%. Thus spatial DNA heterogeneity distribution of diploid cells seems to quantitate probable genetic instability as a function of clinical evolution such as tumour recurrence, and suggests the possible presence of aneuploid stemlines in a heterogeneous tumour, even if a diploid DNA histogram is observed in a single sample. From standardized C1 and C2 canonical discriminant function coefficients, a DNA heterogeneity index (2c-HI) is proposed to characterize diploid cells providing a descriptive and predictive discriminant factor for solid tumour behaviour.

[Role of macrophages in papillomavirus infections]. / Rôle des macrophages dans les infections a papillomavirus.

Papillomaviruses have naturally a strict tropism to epithelial cells in which they replicate during the cell differentiation. There is no histological evidence of any inflammatory reaction. No leucocyte recruitment is observed and thus, the role of macrophages during the early infectious process in the epithelium remains unknown. This silent, subacute or chronic infectious disease is characterized fundamentally by a dual pathogenic process, including an infectious process leading to the production of infective virus particles during the differentiation of infected epithelial cells, on the one hand, and an oncogenic process due to interactions of viral oncogenes with host cell regulatory proteins after integration of the virus to the cellular genome. The role of activated macrophages on the oncogenic process is clearly established. They contribute to regulate negatively the transcription of the non structural E6 E7 viral oncogenes and have cytotoxic effects to transformed cells by a direct intercellular contact without evidence of an effect due to a soluble factor such as tumor-necrotizing factor (TNF). Macrophages have, hence, a prominent role as cellular effectors of protective immunity against lesions due to papillomaviruses and particularly against the oncogenic process complicating these infections.

[An alternative to immunofluorescence on frozen tissue for the diagnosis of sub-epidermal auto-immune bullous dermatosis]. / Une alternative à l'immunofluorescence sur tissu congelé pour le diagnostic des dermatoses bulleuses auto-immunes sous-épidermiques.

The diagnosis of subepidermal auto-immune bullous dermatosis is usually based on immunofluorescence which demonstrates IgA, IgG, IgM or C3 on dermo-epidermal junction. However in some instance, this technique could not be performed. Here we report an alternative to immunofluorescence. It is a preembedding technique of immunolabelling using horseradish peroxidase, on fresh tissue without any freezing, before an inclusion in wax. So the observation of the labelling is possible with a standard microscope. This technique has been applied to 7 patients with various sub-epidermal auto-immune bullous dermatosis whose diagnosis was confirmed elsewhere by immunoelectron microscopy. It was as specific and sensitive than immunofluorescence whatever the bullous dermatosis.

[Superficial cancer of the stomach]. / Les cancers superficiels de l'estomac.

The authors report 17 cases of superficial carcinomas collected amongst a series of 100 gastric carcinomas. Three principal points are analysed: clinical picture, diagnostic and treatment. Clinical problems indicate the multiplicity of classifications used and their sometimes incomplete nature. This led the authors in 1970 to develop a classification with 4 parameters, derived from the T N M classification and from that of Gutmann. The diagnostic problems raises yet again for these superficial carcinomas the need for routine screening which is the only way to increase early diagnoses, and hence the effectiveness of treatment. The therapeutic problem is concerned with the discussion of the limitations of wide excision as a matter of principle.