Alexandros Daponte

Primary Screening for Cervical Cancer Based on High-Risk Human Papillomavirus (HPV) Detection and HPV 16 and HPV 18 Genotyping, in Comparison to Cytology

Objectives The objective of the present study is to assess the performance of a high-risk human papillomavirus (HR-HPV) DNA test with individual HPV-16/HPV-18 genotyping as a method for primary cervical cancer screening compared with liquid-based cytology (LBC) in a population of Greek women taking part in routine cervical cancer screening. Methods The study, conducted by the “HEllenic Real life Multicentric cErvical Screening” (HERMES) study group, involved the recruitment of 4,009 women, aged 25–55, who took part in routine cervical screening at nine Gynecology Departments in Greece. At first visit cervical specimens were collected for LBC and HPV testing using the Roche Cobas 4800 system. Women found positive for either cytology or HPV were referred for colposcopy, whereas women negative for both tests will be retested after three years. The study is ongoing and the results of the first screening round are reported herein. Results Valid results for cytology and HPV testing were obtained for 3,993 women. The overall prevalence of HR-HPV was 12.7%, of HPV-16 2.7% and of HPV-18 1.4%. Of those referred for colposcopy, cervical intraepithelial neoplasia grade 2 or worse (CIN2+) was detected in 41 women (1.07%). At the threshold of CIN2+, cytology [atypical squamous cells of undetermined significance (ASC-US) or worse] and HPV testing showed a sensitivity of 53.7% and 100% respectively, without change between age groups. Cytology and HPV testing showed specificity of 96.8% and 90.3% respectively, which was increased in older women (≥30) in comparison to younger ones (25–29). Genotyping for HPV16/18 had similar accuracy to cytology for the detection of CIN2+ (sensitivity: 58.5%; specificity 97.5%) as well as for triage to colposcopy (sensitivity: 58.5% vs 53.7% for cytology). Conclusion HPV testing has much better sensitivity than cytology to identify high-grade cervical lesions with slightly lower specificity. HPV testing with individual HPV-16/HPV-18 genotyping could represent a more accurate methodology for primary cervical cancer screening in comparison to liquid-based cytology, especially in older women.

High-risk human papillomavirus DNA test and p16INK4a in the triage of LSIL: A prospective diagnostic study

OBJECTIVE The detection of high-grade cervical intraepithelial neoplasia (CIN) amongst patients with low-grade cytology (LSIL) is challenging. This study evaluated the role of high-risk HPV (HR-HPV) DNA test and p16(INK4a) immunostaining in identifying women with LSIL cytology at risk of harboring CIN2 or worse (CIN2+) and the role of p16(INK4a) in the triage of a population of HR-HPV positive LSIL. METHODS We conducted a prospective study including women with LSIL cytology. Detection of HR-HPV was carried out by means of a polymerase chain reaction based assay. p16(INK4a) immunostaining was performed using the Dako CINtec cytology kit. All patients had colposcopically directed punch biopsies or large loop excision of the transformation zone of the cervix. The endpoint was detection of a biopsy-confirmed CIN2+. RESULTS A series of 126 women with LSIL cytology were included. HR-HPV test had sensitivity 75% and specificity 64% for an endpoint of CIN2+. p16(INK4a) had significantly higher specificity of 89% (p=0.0000) but low sensitivity of 42%. The role of p16(INK4a) immunostaining in the triage of LSIL positive for HR-HPV was also evaluated. p16(INK4a) triage had 70% positive predictive value (PPV); however, this was not significantly higher than the PPV (56%) of HR-HPV test alone (p=0.4). CONCLUSIONS The results indicate that HR-HPV or p16(INK4a) cannot be used as solitary markers for the assessment of LSIL. The addition of p16(INK4a) immunostaining led to an increase in HR-HPV specificity; however, the biomarker needs to be assessed further to establish its role as an adjunct test in the triage of LSIL.

Alterations in human papillomavirus-related biomarkers after treatment of cervical intraepithelial neoplasia

OBJECTIVE This study aims to assess the alterations in various HPV-related biomarkers 6 months post-treatment and how these relate to various risk factors and individual characteristics; their role for the prediction of treatment failure was also evaluated. MATERIAL AND METHODS DESIGN Prospective observational study. POPULATION Women planning to undergo treatment for cervical intraepithelial neoplasia. INTERVENTION A liquid-based cytology sample was taken pre-operatively. This was tested for HPV genotyping, Nucleic Acid Sequence Based Amplification, flow cytometric evaluation and p16 immunostaining. A repeat LBC sample was obtained 6 months post-treatment and was tested for the same biomarkers. OUTCOMES The alterations of the biomarkers 6 months post-treatment were recorded. Their relation to individual characteristics and risk factors (age, smoking, sexual history, use of condom, CIN grade, excision margin status, crypt involvement) as well as their role for the prediction of residual/recurrent disease were assessed. ANALYSIS The accuracy parameters (sensitivity, specificity, positive and negative predictive value and the likelihood ratios) of each biomarker for the prediction of recurrent/residual CIN were calculated. RESULTS A total of 190 women were recruited. All biomarkers had significantly higher negativity rates post-treatment compared to pre-treatment ones. Multivariate analysis demonstrated that consistent condom use post-treatment significantly reduces the high-risk HPV positivity rates in comparison to no use (OR=0.18; 95% CI: 0.09-0.38). Sensitivity and specificity for all high risk HPV DNA testing were 0.5/0.62, respectively; the relevant values for only type 16 or 18 DNA typing were 0.5/0.92, for NASBA 0.5/0.94, for flow 0.5/0.85 and for p16 0.25/0.93. CONCLUSION CIN treatment reduces positivity for all HPV-related biomarkers. Consistent condom use significantly reduces high-risk HPV positivity rates. More cases of treatment failures are required in order to specify whether different combinations of HPV-related biomarkers could enhance the accuracy of follow up, possibly in the form of a Scoring System that could allow tailored post-treatment surveillance.

DNA methylation profiles in ovarian cancer: Implication in diagnosis and therapy (Review)

Genetic alterations alone cannot account for the complexity of ovarian cancer. The potential reversibility of epigenetic mechanisms makes them attractive candidates for the prevention and/or treatment of ovarian carcinoma. Detection of the epigenetic signature of each cancer cell may be useful in the identification of candidate biomarkers for disease detection, classification and monitoring and may also facilitate personalized cancer treatment. In ovarian cancer, in addition to other non-gynaecological cancers, two opposite epigenetic phenomena occur. The first involves an overall global decrease in DNA methylation of heterochromatin leading to demethylation of several oncogenes, while the second involves specific CpG island hypermethylation associated with the promoters of tumor suppressor genes. Early studies focused on the methylation patterns of single genes associated with tumorigenesis. However, newer genome-wide methods have identified a group of genes whose regulation is altered by DNA methylation during ovarian cancer progression.

Soluble fms-Like Tyrosine Kinase-1 (sFlt-1) and Serum Placental Growth Factor (PlGF) as Biomarkers for Ectopic Pregnancy and Missed Abortion

CONTEXT Diagnosis of early pregnancy failure is hampered by the lack of reliable serological markers. OBJECTIVE We assessed whether a single serum measurement of placental growth factor (PlGF) and the soluble Flt-1 (sFlt-1) receptor of vascular endothelial growth factor at 6-8 wk gestation could differentiate failed pregnancies, whether ectopic pregnancies (EP) or missed abortions (MA), from healthy intrauterine pregnancies (IUP). DESIGN AND SETTING We conducted a prospective clinical study at a tertiary university hospital between January 2009 and February 2011. PATIENTS A total of 78 consecutive patients (38 EP, 40 MA) with failed early pregnancy and 50 IUP (control group) participated in the study. INTERVENTION(S) Determination of serum PlGF and sFlt-1 has been carried out by ELISA. Gene expression of PlGF and Flt-1 in trophoblasts was performed by RT-PCR. MAIN OUTCOME MEASURE(S) We investigated whether a single, combined serum measurement of the above markers could contribute to the differential diagnosis. RESULTS PlGF and sFlt-1 concentration was lower in both EP (mean, 14.60 ± 3.42/178.16 ± 76.03 pg/ml) and MA (mean, 16.25 ± 4.73/399.42 ± 337.54 pg/ml) compared to IUP (mean, 21.64 ± 5.68/1390.32 ± 655.37 pg/ml). sFlt-1 (P = 0.033) and sFlt-1/PlGF ratio (P = 0.029) but not PlGF had the ability to discriminate MA from EP. Compared to women with viable IUP, mRNA gene expression levels of PlGF and Flt-1 were considerably lower in women with MA and in women with EP. CONCLUSIONS Combined measurement of sFlt-1 and PlGF levels can differentiate normal from failed pregnancies, whereas sFlt-1 as well as sFlt-1/PlGF ratio can also discriminate EP from MA. PlGF and Flt-1 gene expression in trophoblasts from women with EP and MA appears impaired.

Activin A and Follistatin as Biomarkers for Ectopic Pregnancy and Missed Abortion

Activin A as a predictor of pregnancy failure has been the focus of heated debate, but the value of a combined activin A and follistatin (FS) measurement in serum to predict pregnancy failure has not been reported yet. We assessed whether a single serum measurement of the two physiological antagonists at 6–8 weeks gestation could differentiate ectopic pregnancies (EP) or missed abortions (MA) from healthy intrauterine pregnancies (IUP). activin A concentrations were significantly lower in women with EP (n = 30, median value of 264 pg/mL) and women with MA (n = 30, median value of 350 pg/mL) compared to IUP (n = 33, median value of 788 pg/mL); P < 0.001. At a threshold value of 505 pg/mL, activin A had 87.9% sensitivity and 100% specificity and negative predictive value of 0.974 for discriminating an ectopic pregnancy from viable pregnancies. FS was able to discriminate IUP from EP (ROC curve P < 0.001) as was their ratio (ROC curve P = 0.008), but was unable to discriminate a MA from an EP. In EP, activin A did not correlate with beta HCG levels. The present findings support the thesis that activin A or FS could be considered promising biomarkers for the discrimination between an IUP and a failed pregnancy (MA or EP).

Serum interleukin-1β, interleukin-8 and anti-heat shock 60 Chlamydia trachomatis antibodies as markers of ectopic pregnancy

Anti-Chlamydial trachomatis (anti-CT) responses, particularly anti-heat shock 60 (Hsp60), antibodies confer a higher risk of ectopic pregnancy. With emerging evidence supporting the pivotal role of interleukin-1β (IL-1β) and IL-8 in the immunopathogenesis of CT-specific tubal obstruction, we determined anti-CT Hsp60 antibody reactivity and serum concentrations of IL-1β and IL-8 in failed pregnancies consisting of 30 consecutive ectopic pregnancies and 30 missed abortions, with 32 viable intrauterine pregnancies tested as normal controls. ELISAs were utilised to measure IgA or IgG anti-CT major outer membrane outer protein (MOMP) antibodies, IgG anti-CT Hsp60 antibodies and IL-1β and IL-8. IgG anti-CT Hsp60 antibodies were more prevalent in ectopic pregnancy cases (43.3%, 13/30) than in intrauterine pregnancies (16%, 5/32, p=0.016). All 13 ectopic pregnancy anti-CT Hsp60-positive cases had anti-CT MOMP antibodies. CT-specific antibodies were more frequent in merged ectopic pregnancy and missed abortions cases (35%, 21/60) than in intrauterine pregnancies (16%, p=0.049). The median (range) levels of IL-1β in ectopic pregnancy, missed abortions and normal intrauterine pregnancies were 1.74 (0.2-8.7), 1.14 (0.2-16) and 1.22 (0.2-16.2) pg/ml, respectively (p>0.05, for all). Serum IL-8 levels were comparable amongst groups: ectopic pregnancy (median [range]: 25.1 [18.3-1000]); missed abortions (32.9 [15.39-1000]); and intrauterine pregnancies (25.11 [18.3-1000] pg/ml). Anti-CT antibody-positive ectopic pregnancy had significantly lower IL-1β levels (1.29 [0.2-2.93]) pg/ml than sero-negative ectopic pregnancy cases (2.09 [1.10-8.70]) pg/ml, (p=0.022), but IL-8 did not differ. Our data demonstrate that anti-CT Hsp60 immunity is a predominant feature of ectopic pregnancy. We conclude that neither IL-1β nor IL-8 can be considered markers of failed pregnancy, although lower levels of the former cytokine are associated with CT-related ectopic pregnancy.

Angiopoietin-1 and angiopoietin-2 as serum biomarkers for ectopic pregnancy and missed abortion: A case–control study

BACKGROUND A case-control study to evaluate whether a single serum measurement of angiopoietin-1 (ANG-1) and angiopoietin-2 (ANG-2) at 6-8 weeks gestation can differentiate failed pregnancies, whether ectopic pregnancies (EP) or missed abortions (MA), from healthy intrauterine pregnancies (IUP). INTERVENTION(S) Serum and tissue mRNA determination of ANG-1 and ANG-2 levels by ELISA and RTPCR, from 60 (30 EP and 30 MA) patients with failed early pregnancy and 33 IUPs. RESULTS ANG-1 and ANG-2 concentrations and their ratio are lower in EP (median, 689 and 302 pg/ml, respectively) and MA cases (median, 810 and 402 pg/ml, respectively) compared to IUP (median, 963 and 1477 pg/ml, respectively) (p<0.05, for all). Unlike ANG-2, serum ANG-1 discriminates an EP from a MA (p=0.011). Trophoblastic ANG-1 mRNA expression levels are lower in EP compared to MA and IUP (p<0.05), while ANG-2 mRNA is higher in EP and MA than in IUP (p<0.05). CONCLUSIONS A single measurement of serum ANG-1 and ANG-2 at 6-8 weeks of gestation designate the outcome of a pregnancy, as their levels are significantly decreased in failed than normal pregnancies. Serum ANG-1 showed potential to discriminate MA from EP.

The role of cytokines and hot flashes in perimenopausal depression

BackgroundAn imbalance in the production of proinflammatory and anti-inflammatory cytokines may play a role in the pathophysiology of perimenopausal depression. The aim of this study was to examine serum levels of the proinflammatory cytokines interleukin 6 (IL-6) and tumor necrosis factor α (TNFα), and the anti-inflammatory cytokine IL-10, in perimenopausal women suffering from depression. Furthermore, to assess whether serum cytokine levels are associated with the presence of hot flashes or the use of selective serotonin reuptake inhibitors (SSRIs). We also evaluated the possible association of hot flashes and perimenopausal depression.MethodsSerum samples from 65 perimenopausal women, 41 with depression and 24 without depression, were assessed for serum IL-6, TNFα and IL-10 by conventional enzyme-linked immunosorbent assays. Depression was evaluated by the 17-item Hamilton Depression Rating Scale (HAM-D 17) and a psychiatric interview. The presence and severity of hot flashes were examined using the Menopause Rating Scale (MRS).ResultsSerum levels cytokines did not differ between depressed women and normal controls. Serum levels of cytokines did not change significantly in depressed women with hot flashes or in depressed women treated with SSRIs. Hot flashes were strongly associated (P < 0.0001) with perimenopausal depression.ConclusionThe study supports the hypothesis that perimenopausal depression is not characterized by increased proinflammatory cytokines and decreased anti-inflammatory cytokines. Women with perimenopausal depression suffer from more severe and more frequent hot flashes than women without perimenopausal depression.

Fascin in ovarian epithelial tumors

Fascin contributes to the formation of actin-based protrusions involved in cell migration. Fascin has emerged as a prognostic marker in some carcinomas. We examined ovarian neoplasms to check any correlation between fascin expression and established clinicopathologic parameters. Fascin immunoreactivity was semiquantitavely scored in 100 ovarian tumors (62 carcinomas, 15 borderline tumors and 23 cystadenomas). Double staining for fascin and Ki-67 was performed in selected carcinomas. Western Blotting was done in frozen samples. Fascin immunoreactivity was highest in carcinomas, lowest in cystadenomas and intermediate in borderline tumors; these results were in accordance with those from Western blotting analysis. Fascin was statistically increased in carcinomas of advanced stage and in serous carcinomas. It was also increased in metastatic foci and in tumor foci with lower Ki-67 labeling. We conclude that in ovarian tumors fascin is associated with certain features of increased tumor aggressiveness. Future studies could determine if fascin may become a routinely helpful marker in gynecological pathology or clinical oncology.