Cecilia Bussani

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 Spearmans 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.

DNA methylation of HOXA10 in eutopic and ectopic endometrium

STUDY QUESTION Does the methylation status of the promoter region of the HOXA10 gene differ in eutopic and ectopic endometrium? SUMMARY ANSWER The eutopic endometrium in women with endometriosis is significantly more methylated when compared with controls. WHAT IS KNOWN ALREADY Expression of the HOXA10 gene, which is important for successful implantation, is reduced in women affected by endometriosis. STUDY DESIGN, SIZE AND DURATION A pilot study was carried out including 18 women admitted for surgery for endometriosis-related pain (cases) and 12 women admitted for surgery because of non-endometriotic disease (control). Sample collection and analysis were performed between November 2010 and July 2013. PARTICIPANTS/MATERIALS, SETTING, METHODS Endometrial tissue (eutopic and ectopic) underwent sodium bisulfite DNA modification, PCR amplification of two regions of the HOXA10 promoter and pyrosequencing analysis. MAIN RESULTS AND THE ROLE OF CHANCE The eutopic endometrium of women with endometriosis was significantly more methylated compared with endometrium from the control group (sequence 1: 8.68% in cases and 6.25% in the control group: P = 0.037, sequence 2: 11.89% in cases and 9.25% in the control group: P = 0.032). The eutopic endometrium was significantly more methylated than the ectopic tissue in patients with endometriosis (mean difference -3.6 sequence 1: P = 0.001 and -6.0 sequence 2: P = 0.0001). LIMITATIONS, REASONS FOR CAUTION The study had a limited sample size and the fertility status of the majority of patients in our study was unknown. WIDER IMPLICATIONS OF THE FINDINGS Our data regarding methylation state of the ectopic tissues contribute to a better etiopathologic understanding of endometriosis. STUDY FUNDING/COMPETING INTERESTS No external funding was either sought or obtained for this study. The authors have no conflicts of interests to declare.

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.

Prenatal Diagnosis of Common Aneuploidies in Transcervical Samples Using Quantitative Fluorescent-PCR Analysis

AbstractAim: 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.

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.

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.

Use of the Quantitative Fluorescent-PCR Assay in the Study of Fetal DNA from Micromanipulated Transcervical Samples

AbstractAim: 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.

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.

Methylation of the HOXA10 homeobox gene promoter is associated with endometrial cancer: A pilot study

Methylation in the promoter region represents an epigenetic mechanism that silences expression of various homeobox genes in cancers. We compare the methylation profile of HOXA10 promoter gene in 19 histologically proven endometrioid cancers and 27 normal endometrial tissues. Endometrial cancer tissue displays significantly higher methylation status in HOXA10 gene promoter than normal tissue, suggesting a possible role of epigenetic changes in HOXA10 gene regulation in tumorigenesis. Further studies in human tissue and cell lines are necessary to validate these preliminary results and to investigate HOXA10 expression according to methylation status in endometrial cancer.

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

This study aims to estimate whether chorionic villous sampling (CVS) causes a significant increase of cell‐free fetal DNA (cffDNA) in maternal circulation.