HPV genotype distribution and age correlation in a selected Italian population undergoing conization.

BACKGROUND: Detection and genotyping of human papillomavirus (HPV) has gained increasing importance in cervical cancer prevention and treatment of cervical intraepithelial neoplasia (CIN). This study aims to determine the HPV type distribution in cervical specimens obtained from women diagnosed with CIN. We evaluated in a selected Italian population the distribution of HPV genotypes. METHODS: Cervical samples were collected from women undergoing laser CO2 conization for high grade at Colposcopic Laser Surgery Unit of the Careggi University Hospital and at the Colposcopy Service of Local Health Unit Toscana Centro in Florence, Italy, between September 2014 and February 2017. HPV genotyping was performed using the LINEAR ARRAY® HPV Genotyping Test. RESULTS: Three hundred and six patients were enrolled. HPV infection was detected on 244 samples (79.7%). A different rate of mono- and poly-infections was observed, with higher poly-infection rates in younger women. Moreover, depending on different age groups (clustered in 5-years interval from 22 to 69 years old) significant different distribution of HPV was fund as genotype, phylogenetic type and cancer-related risk. CONCLUSIONS: Our results suggest that some physiological conditions (i.e. menopause), could influence selection and clearance of specific HPV genotypes. The results of this study represent the basis for supporting the HPV genotyping as clinical tool providing benefits in the management of women with high CIN grade.

High grade cervical intraepithelial neoplasia positive biopsy: the importance of accurate pre-operative workup.

BACKGROUND: In cervical cancer screening programs, women with abnormal cytology and confirmation by biopsy are referred for colposcopy for histological evaluation. METHODS: We characterized the presence and the genotype of HPV by Linear Array HPV genotyping assay in cytological samples collected from about 400 women undergoing conization, with reported high CIN grade after biopsy. RESULTS: The most prevalent genotype was HPV 16, with an increasing presence depending on the severity of the CIN and with the highest incidence in the 26-35 age range. In the group of younger women (<25) we found the highest percentage of CIN3 (39.3%) and the lowest of CIN1 (17.9%). An increase of CIN1 with increasing age was observed. A different distribution of HPV presence was observed depending on CIN grade (P<0.001): CIN1 HPV negative samples were 46.3%, CIN2: 5.8% and CIN3: 1.4%. Interesting, in the analyzed cohort, we observed the presence of 30% of CIN1. Moreover, within CIN1, 85% of them were associated to negative HPV detection, this observation suggested that the detection of HPV presence may be useful to identify low CIN grade that should be reconsidered for surgical treatment. CONCLUSIONS: These findings suggest implementing the protocol for the management of women with high risk precancer lesions, with a further HPV test before surgical treatment. The evaluation of HPV presence and genotype before conization might represent a useful tool in reducing or postpone the conization treatment.

Indication of prophylactic vaccines as a tool for secondary prevention in HPV-linked disease.

PURPOSE: To determine whether quadrivalent HPV vaccination is effective in reducing recurrent disease in women with a previous history of HPV disease. METHODS: All women under 45 years of age treated for HPV-linked disease and with negative HPV test, cytology and colposcopy 3 months after treatment were enrolled. Women were randomly assigned into two groups: a group that received HPV vaccine post treatment and a group that was only submitted to follow-up. Follow-up was performed every 6 months for a duration of at least 3 years. Kaplan-Meier curve was used to estimate the overall disease-free survival during the follow-up period. Statistical analysis was performed by Fisher's exact test. RESULTS: From November 2013 to October 2014, we enrolled a total of 178 women at Careggi University Hospital in Florence and at Azienda USL in Massa Carrara. 12 out of 89 patients in the non-vaccination group recurred (13.5%), while 3 out of 89 patients in the vaccination group recurred (3.4%). The Kaplan-Meier curves showed a statistically difference in the log rank test (p = 0.0147) for the overall disease-free survival in the study groups during follow-up. The rate of recurrence was significantly higher in the non-vaccination group, with a p = 0.0279 by Fisher exact test. CONCLUSION: The introduction of anti-HPV vaccination during the follow-up post treatment for HPV-linked disease is recommended to reduce the risk of recurrence. The clinical implication of this could be very important to influence post-treatment management of HPV disease.

Hypermethylation of HOXA10 gene in mid-luteal endometrium from women with ovarian endometriomas.

A decrease in HOXA10 gene expression in eutopic mid-secretory endometrium has been found in women with endometriosis-associated infertility. Promoter hypermethylation of HOXA10 is thought to be the leading mechanism for epigenetic gene regulation in patients with endometriosis. In our series we documented significantly higher HOXA10 promoter methylation levels in women with ovarian endometriomas than in healthy controls during the mid-luteal phase. Development of epigenetic-based strategies for non-surgical treatment of infertility related to ovarian endometriomas could be an attractive field of research in the coming years.

Cell-free fetal DNA in maternal circulation after chorionic villous sampling.

OBJECTIVE: This study aims to estimate whether chorionic villous sampling (CVS) causes a significant increase of cell-free fetal DNA (cffDNA) in maternal circulation. METHOD: Fifty pregnant women with singleton pregnancy were recruited prior to CVS. Maternal peripheral blood was collected before and after CVS. A methylation-sensitive restriction enzyme digestion was used to select the placental-derived hypermethylated promoter of the RASSF1A gene in maternal plasma, thus differentiating cffDNA from mother's cell-free DNA (cfDNA), where the RASSF1A gene is normally hypomethylated. Total cfDNA and cffDNA amounts were compared before and after CVS in each patient. Data were compared using the Student t-test. RESULTS: No significant difference before and after CVS was found between the following: (i) total cfDNA concentration in plasma (p = 0.695); (ii) cffDNA concentration in plasma (p = 0.612); and (iii) percentage of fetal DNA in plasma (p = 0.835). After dividing the cases on the basis of the sex of the fetus, maternal age, gestational age, number of pregnancies, position of the placenta, and presence of trisomy of the fetus, no difference in fetal and total DNA concentrations before and after CVS was observed. CONCLUSION: The CVS does not seem to significantly disrupt the maternal-placental interface, as no significant increase of cffDNA in maternal plasma following CVS was observed.

The zinc finger gene ZIC2 has features of an oncogene and its overexpression correlates strongly with the clinical course of epithelial ovarian cancer.

PURPOSE: Epithelial ovarian tumors (EOT) are among the most lethal of malignancies in women. We have previously identified ZIC2 as expressed at a higher level in samples of a malignant form (MAL) of EOT than in samples of a form with low malignant potential (LMP). We have now investigated the role of ZIC2 in driving tumor growth and its association with clinical outcomes. EXPERIMENTAL DESIGN: ZIC2 expression levels were analyzed in two independent tumor tissue collections of LMP and MAL. In vitro experiments aimed to test the role of ZIC2 as a transforming gene. Cox models were used to correlate ZIC2 expression with clinical endpoints. RESULTS: ZIC2 expression was about 40-fold in terms of mRNA and about 17-fold in terms of protein in MAL (n = 193) versus LMP (n = 39) tumors. ZIC2 mRNA levels were high in MAL cell lines but undetectable in LMP cell lines. Overexpression of ZIC2 was localized to the nucleus. ZIC2 overexpression increases the growth rate and foci formation of NIH3T3 cells and stimulates anchorage-independent colony formation; downregulation of ZIC2 decreases the growth rate of MAL cell lines. Zinc finger domains 1 and 2 are required for transforming activity. In stage I MAL, ZIC2 expression was significantly associated with overall survival in both univariate (P = 0.046) and multivariate model (P = 0.049). CONCLUSIONS: ZIC2, a transcription factor related to the sonic hedgehog pathway, is a strong discriminant between MAL and LMP tumors: it may be a major determinant of outcome of EOTs.

Quantitative variation analysis of fetal DNA in maternal plasma samples collected before and after amniocentesis.

AIMS: The aim of this study was to evaluate possible procedure-related variations in the levels of cell-free fetal DNA (fDNA) in maternal plasma of women undergoing genetic amniocentesis. MATERIALS AND METHODS: Blood samples were collected at 16-18 weeks' gestation from 33 pregnant women attending the Fetal Medicine Unit for genetic amniocentesis. For each woman, two blood samples were obtained: the first immediately before amniocentesis and the second one 15 min after the procedure. A real-time quantitative polymerase chain reaction assay, using primers for SRY and beta-globin genes, was used to assess fDNA concentrations in maternal plasma. A Wilcoxon signed-rank test was used for statistical analysis. RESULTS: The karyotype on cultured amniocytes showed that 15 out of 33 women had a male fetus. Real-time quantitative polymerase chain reaction results, on maternal plasma sample pairs from known male pregnancies, showed no significant variations of fDNA correlated to amniocentesis (P=0.394). CONCLUSIONS: Our preliminary study suggests that amniocentesis, although invasive, could be associated with minimal, if any, disruption at the fetal-maternal interface, as revealed by the lack of substantial modifications of fDNA levels in maternal circulation.

Preoperative assessment of HER-2/neu status in breast carcinoma: the role of quantitative real-time PCR on core-biopsy specimens.

OBJECTIVES: Knowledge of HER-2/neu status is mandatory to identify breast cancer patients amenable to trastuzumab treatment. We evaluated the diagnostic performance of quantitative real-time polymerase chain reaction (qRT-PCR) in the preoperative determination of HER-2/neu status in breast cancer, using core biopsy material. METHODS: In a prospective series, qRT-PCR was performed on fresh core biopsy specimens taken preoperatively in 87 patients with breast carcinoma. Cases with qRT-PCR ratio > or = 2.0 were considered to have HER-2/neu amplification. The results of RT-PCR analysis were compared with those of the standard immunohistochemistry (IHC) and Fluorescence in situ hybridization (FISH) methods. Cases with IHC 3+ or with IHC 2+ and FISH showing amplification were considered HER-2/neu positive. All other cases were considered HER-2/neu negative. RESULTS: qRT-PCR showed HER-2/neu amplification in 13 cases (14.9%), while the standard IHC-FISH combined approach identified 17 HER-2/neu-positive cases (19.5%). Overall, there was concordance between methods in 83 of 87 patients (95.4%). The Spearman's rho correlation coefficient was 0.851; p<0.001. The diagnostic performance for preoperative diagnosis of HER-2/neu status using RT-PCR on core biopsy specimens as compared to standard approach was as follows: sensitivity 76.5%; specificity 100%; positive predictive value 100%; negative predictive value 94.6%. CONCLUSIONS: Quantitative RT-PCR determination of HER-2/neu status from core biopsy specimens provided results comparable to those given by the standard IHC and FISH methods. The use of qRT-PCR on core biopsy material may represent a very useful and easy tool to enhance early identification of HER-2/neu-positive breast cancer patients who, possibly can benefit from trastuzumab treatment.

The presence of trophoblastic cells in intrauterine lavage samples: lack of correlation with maternal and obstetric characteristics.

OBJECTIVES: To investigate the correlation between maternal, obstetric and sample characteristics and the quality (i.e. yield of trophoblastic cells) of intrauterine lavage (IUL) samples. METHODS: We collected 202 IUL samples from women scheduled for first trimester termination of pregnancy (TOP). Trophoblastic cells were isolated from IUL samples and used for DNA analysis by a multiplex quantitative fluorescent polymerase chain reaction (QF-PCR) assay. A multivariate logistic regression analysis was performed, and a p<0.05 was considered statistically significant. RESULTS: The presence of trophoblastic cells in IUL samples was documented in 151/202 cases (74.7%). Blood contamination of IULs was the only characteristic to positively correlate with the presence of trophoblasts (p=0.039; OR: 1.99; 95% CI: 1.03-3.82). CONCLUSIONS: The correlation between the presence of contaminating blood and trophoblastic cells would indirectly confirm the hypothesis that IUL might act as a mini-CVS. The high yield rate of trophoblasts irrespective of maternal characteristics and past obstetric history would support the clinical use of this sampling technique, provided that its safety is clearly defined.

PCR detection rates of high risk human papillomavirus DNA in paired self-collected urine and cervical scrapes after laser CO2 conization for high-grade cervical intraepithelial neoplasia.

OBJECTIVE: To compare the PCR detection rates of high risk human papillomavirus DNA in self-collected urine and cervical scrapes during follow-up of patients treated for HG-CIN by laser CO2 conization. PATIENTS AND METHODS: 52 women who submitted to laser conization for HG-CIN were enrolled into this prospective follow-up study receiving liquid-based cytology and HR-HPV testing by PCR assay on self-collected urine and cervical scrapes before and at 3, 6 and 12 months after treatment. Diagnostic accuracy and predictive values for treatment failure were evaluated for both urinary and cervical HPV testing and follow-up cytology. RESULTS: 3 cases (5.8%) of recurrent HG-CIN occurred during follow-up. Positive margins and HR-HPV persistence resulted to significant risk factors for recurrence (p=0.01). The overall concordance on HR-HPV detection between paired urine and cervical samples was 96.6% and discord trend between agreement rates during follow-up were excluded by overall fixed-effect index (OR 1.03; 95% CI 0.62-1.70). No difference was observed comparing the three- and six-month cumulative sensitivity and NPV for recurrent disease of urinary and cervical HPV detections, with an increase of 5.6% in specificity associated with urinary testing. CONCLUSIONS: PCR detection of HR-HPV in paired urine and cervical samples during follow-up revealed an excellent concordance, suggesting a potential equivalent role of the two methods within post-treatment follow-up. In our experience HPV testing on self-collected urine was more sensitive than cytology and more specific than cervical HPV detection to predict treatment failure. Larger studies are needed to definitively establish the role of urine-based HPV testing during follow-up.

Prenatal diagnosis of common aneuploidies in transcervical samples using quantitative fluorescent-PCR analysis.

AIM: The aim of this study was to test the feasibility of diagnosing common fetal chromosomal aneuploidies using quantitative fluorescent (QF)-PCR on transcervical cell (TCC) samples collected in the first trimester of pregnancy by means of intrauterine lavage (IUL). METHODS: A total of 181 TCC samples were retrieved from pregnant women between 5 and 12 weeks of gestation, immediately before elective termination of pregnancy, at which time corresponding placental tissue and maternal blood specimens were also obtained. Isolation of trophoblastic cells by micromanipulation was attempted in all TCC samples. Micromanipulated specimens were analyzed by multiplex QF-PCR, including short tandem repeats for the chromosomes X, Y, 21, 18, and 13. RESULTS: The micromanipulation was successful in 152 of 181 cases (84.8%) where chorionic villous filaments and/or cell clumps of seeming trophoblastic origin could be isolated. All 152 samples were tested by QF-PCR analysis and peaks of paternal origin could be documented in all cases. Two cases of trisomy 21 and two cases of monosomy X0 were detected by means of QF-PCR assay, in accordance with the results obtained in corresponding placental samples. CONCLUSION: This study provides evidence that the use of multiplex QF-PCR amplification of selected microsatellites could be applied to micromanipulated TCC samples and in particular to IUL samples, which often contain trophoblastic cells, for the detection of chromosomal aneuploidies. The approach described in this study appears, therefore, a very promising tool toward non-invasive prenatal genetic diagnosis in the early stage of gestation.

Fetal cells in a transcervical cell sample collected at 5 weeks of gestation.

Transcervical cell (TCC) sampling is being investigated as a promising method for obtaining fetal cells for prenatal genetic diagnosis. The present case report describes the identification of fetal cells by both fluorescent in situ hybridisation (FISH) and quantitative fluorescent polymerase chain reaction (QF-PCR) analyses in a TCC sample collected by intrauterine lavage at 5 + 0 weeks. This finding underscores the possible relevance of TCC sampling for extremely early prenatal genetic diagnosis.

Comparison of two techniques for transcervical cell sampling performed in the same study population.

OBJECTIVES: The aim of this study was to evaluate and compare the presence of fetal cells in transcervical cell (TCC) samples collected in the first trimester of pregnancy by two different procedures [mucus collection and intrauterine lavage (IUL)], performed consecutively in the same subjects scheduled for elective termination of pregnancy (TOP). METHODS: A total of 126 mucus/IUL sample pairs were retrieved from pregnant women immediately before TOP at a gestational age ranging from 7 to 12 weeks; at termination, samples of placental tissue were collected in all cases. All mucus samples were analysed by a polymerase chain reaction (PCR) assay and, in a subset of experiments involving 56 specimens, also by fluorescence in situ hybridization (FISH) procedure. IULs were divided in two aliquots, one for PCR analysis and one for the preparation of FISH slides. All placental tissue samples obtained at termination were analysed by FISH for fetal sexing. The PCR assay for fetal sex determination was performed by using, in a multiplex reaction, primers for SRY (Y chromosome sex-determining region, 738 bp) and HUMARA (human androgen receptor on the X chromosome, 280 bp) genes. The FISH analysis was carried out using direct-labelled commercial probes for X chromosome alpha-satellite (DXZ1, Xp11.1-q11.1, spectrum green) and Y chromosome alpha-satellite (DYZ3, Yp11.1-q11.1, spectrum orange) regions. RESULTS: In samples from known male pregnancies (n = 67), full concordance between IUL and mucus results could be found in 11 cases (16.4%); in 41 cases, Y chromosome material was detected by FISH (n = 2), by PCR (n = 5) or both (n = 34) in IUL samples, but not in the corresponding mucus samples. Y chromosome material was not documented in 10 mucus/IUL sample pairs. In 5 cases, the FISH (n = 2), the PCR (n = 1) or both (n = 2) failed to detect Y chromosome material in IULs, which was detected, however, by PCR in the corresponding mucus samples. Overall, correct sex prediction was achieved in 55/67 IULs (82%) and in 16/67 (23.9%) mucus samples from male pregnancies. In samples from known female pregnancies (n = 56), full concordance between results of IUL/mucus pairs and those on placental samples could be found in 53 cases (94.6%); in 3 cases, Y chromosome material was documented by PCR in mucus samples, but not in the corresponding IULs. Correct sex prediction was therefore achieved in 56/56 IULs (100%) and in 53/56 (94.6%) mucus samples from female pregnancies. CONCLUSION: This study provides evidence that, among TCC sampling techniques, IUL, but not mucus collection, can yield fetal cells in a constant and reliable fashion, which is a basic prerequisite for possible clinical usage. This suggestion had already emerged from some previous investigations but, owing to the study design, differences in study populations can no longer be used to explain the very different and sometimes-conflicting results reported in earlier studies.

Premature ovarian failure and fragile X premutation: a study on 45 women.

OBJECTIVE: The aim of this study was to test for the presence of the fragile X (FRAXA) premutation a group of women with early menopause. STUDY DESIGN: 45 women with idiopathic premature ovarian failure (POF), five with a familial and 40 with a sporadic form, were screened for the presence of FRAXA premutation. A control group of 28 women >45 years, with one or more children and no signs of POF, was also studied. RESULTS: We found three cases of fragile X premutations in women all belonging to the group with sporadic POF. CONCLUSION: Our results seems to confirm previous observations on the non random association between POF and FRAXA premutation.

Use of the quantitative fluorescent-PCR assay in the study of fetal DNA from micromanipulated transcervical samples.

AIM: The purpose of this study was to evaluate the validity of the combined use of micromanipulation and quantitative fluorescent (QF)-PCR assay for the identification of fetal elements in transcervical cell (TCC) samples collected in early pregnancy. METHODS: TCC samples were obtained by intrauterine lavage (IUL) in 113 pregnant women who were between 7 and 12 weeks pregnant before termination of pregnancy. All IUL samples were screened under an inverted microscope, at which time the isolation of fetal cells by micromanipulation was attempted. QF-PCR assay, using 9 small tandem repeat (STR) markers for chromosomes 13, 18, 21, X, and Y, was performed in all specimens to identify fetal cells in TCC samples and the corresponding placental tissue and blood specimens. TCC samples from male fetuses in which either the micromanipulation or QF-PCR analysis were unsuccessful, were studied with fluorescent in situ hybridization (FISH), using probes for X and Y chromosomes. RESULTS: Isolation of supposed fetal material from IUL samples was carried out by means of micromanipulation in 93 cases (82.3%), where discernible chorionic villous filaments or cell clumps of probable trophoblastic origin were present. The QF-PCR analysis was performed in all 93 IUL samples and paternal peaks could be documented in 88 cases (94.6%) thus confirming the presence of fetal cells. Thirteen cases negative to micromanipulation and derived from male fetuses and four male cases not informative with QF-PCR analysis, after micromanipulation, were then tested with FISH assay using probes for sexual chromosomes. In six samples, rare (2-3%) male fetal cells were detected. Considering the combined results obtained from QF-PCR and FISH assays, the overall fetal sexing was correct in 83.2% of cases (94 of 113). CONCLUSION: This study provides evidence that fetal cells are present in a high proportion of IUL samples. Micromanipulation appears to be an extremely efficient method for the isolation of trophoblastic elements. This study also confirms the potential of IUL as a possible alternative to the traditional prenatal diagnostic procedures for the recovery of fetal cells in precocious stage of gestation, and validates the combination of the isolation of such fetal elements by means of micromanipulation and analysis with the QF-PCR assay for the identification of the most frequent prenatal chromosomal aneuploidies.

Detection of fetal DNA in the peritoneal cavity during pregnancy.

There is increasing evidence that fetal cells are commonly shed toward the cervix and in maternal circulation during pregnancy. In this study, a sample of peritoneal fluid was retrieved from a primigravida at 12 weeks' gestation undergoing urgent intervention for the torsion of an adnexal mass; the sample was then analysed by a polymerase chain reaction (PCR) assay using primers for X- and Y-chromosome specific sequences, and Y-derived sequences were identified. The course of pregnancy was then uneventful until term, when the patient delivered a male fetus, thus, supporting the hypothesis of a fetal origin for the Y-derived sequences detected in the peritoneal fluid. Further studies are required in order to confirm these findings and precisely define the origin of these sequences; however, this report seems to provide further evidence of the spreading of fetal cells during gestation and addresses relevant issues as to the possibility of collecting these cells by culdocentesis and intraperitoneal lavage for prenatal diagnosis.