First-in-Human Phase I Clinical Trial of an SFV-Based RNA Replicon Cancer Vaccine against HPV-Induced Cancers.

A first-in-human phase I trial of Vvax001, an alphavirus-based therapeutic cancer vaccine against human papillomavirus (HPV)-induced cancers was performed assessing immunological activity, safety, and tolerability. Vvax001 consists of replication-incompetent Semliki Forest virus replicon particles encoding HPV16-derived antigens E6 and E7. Twelve participants with a history of cervical intraepithelial neoplasia were included. Four cohorts of three participants were treated per dose level, ranging from 5 × 105 to 2.5 × 108 infectious particles per immunization. The participants received three immunizations with a 3-week interval. For immune monitoring, blood was drawn before immunization and 1 week after the second and third immunization. Immunization with Vvax001 was safe and well tolerated, with only mild injection site reactions, and resulted in both CD4+ and CD8+ T cell responses against E6 and E7 antigens. Even the lowest dose of 5 × 105 infectious particles elicited E6/E7-specific interferon (IFN)-γ responses in all three participants in this cohort. Overall, immunization resulted in positive vaccine-induced immune responses in 12 of 12 participants in one or more assays performed. In conclusion, Vvax001 was safe and induced immune responses in all participants. These data strongly support further clinical evaluation of Vvax001 as a therapeutic vaccine in patients with HPV-related malignancies.

DNA methylation markers as triage test for the early identification of cervical lesions in a Chinese population.

Objective strategies are required in cervical cancer screening. We have identified several DNA methylation markers with high sensitivity and specificity to detect cervical intraepithelial neoplasia 2 or worse (CIN2+) in Dutch women. Our study aims to analyze the diagnostic characteristics of these markers in a Chinese cohort. A total of 246 liquid-based cytology samples were included, of which 205 women underwent colposcopy due to an abnormal cytology result (atypical squamous cells of undetermined significance [ASCUS] or worse), while 227 were tested high-risk human papillomavirus (hrHPV) positive. All six individual markers (ANKRD18CP, C13ORF18, EPB41L3, JAM3, SOX1 and ZSCAN1) showed enhanced methylation levels and frequency with increasing severity of the underlying lesion (P ≤ .001). In cytological abnormal women, sensitivity to detect CIN2+ was 79%, 76% and 72% for the three panels (C13ORF18/EBP41L3/JAM3, C13ORF18/ANKRD18CP/JAM3 and ZSCAN1/SOX1, respectively), with a specificity of 57%, 65% and 68%. For the first two panels, these diagnostic characteristics were similar to the Dutch cohort, while for ZSCAN1/SOX1 the sensitivity was higher in the Chinese cohort, but with a lower specificity (both P < .05). In hrHPV-positive samples, similar sensitivity and specificity for the detection of CIN2+ were found as for the abnormal cytology cohort, which were now all similar between both cohorts and non-inferior to HPV16/18 genotyping. Our analysis reveals that the diagnostic performances are highly comparable for C13ORF18/EBP41L3/JAM3 and C13ORF18/ANKRD18CP/JAM3 methylation marker panels in both Chinese and Dutch cohorts. In conclusion, methylation panels identified in a Dutch population are also applicable for triage testing in cervical cancer screening in China.

DNA methylation markers as a triage test for identification of cervical lesions in a high risk human papillomavirus positive screening cohort.

Objective triage strategies are required to prevent unnecessary referrals for colposcopy in population-based screening programs using primary high-risk human papillomavirus (hrHPV) testing. We have identified several DNA methylation markers with high sensitivity and specificity for detection of high-grade cervical intraepithelial neoplasia or worse (CIN2+) in women referred for colposcopy. Our study assessed diagnostic potential of these methylation markers in a hrHPV-positive screening cohort. All six markers (JAM3, EPB41L3, C13orf18, ANKRD18CP, ZSCAN1 and SOX1) showed similar association across histology in the hrHPV-positive cohort when compared to the Dutch cohort (each p > 0.15). Sensitivity for CIN2+ was higher using methylation panel C13orf18/EPB41L3/JAM3 compared to the other 2 panels (80% vs. 60% (ANKRD18CP/C13orf18/JAM3) and 63% (SOX1/ZSCAN1), p = 0.01). For CIN3+ all three methylation panels showed comparable sensitivity ranging from 68% (13/19) to 95% (18/19). Specificity of SOX1/ZSCAN1 panel (84%, 167/200) was considerably higher compared to ANKRD18CP/C13orf18/JAM3 (68%, 136/200, p = 2 × 10-5 ) and C13orf18/EPB41L3/JAM3 (66%, 132/200, p = 2 × 10-7 ). High negative predictive value (NPV) (91-95% and 96-99%) was observed for CIN2+ and CIN3+, for all three methylation panels, while positive predictive value (PPV) varied from 25 to 40% for CIN2+ and 15-27% for CIN3+. Interestingly, 118/235 samples were negative for all six markers (including 106 controls (89.8%), 6 CIN1 (5.1%), 5 CIN2 (4.2%) and 1 CIN3 (0.8%)). Methylation results from both independent cohorts were comparable as well as high sensitivity for detection of cervical cancer and its high-grade precursors in hrHPV-positive population. Our study therefore validates these methylation marker panels as triage test either in hrHPV-based or abnormal cytology-based screening programs.

Prognostic factors for local recurrence of squamous cell carcinoma of the vulva: A systematic review.

BACKGROUND: In patients treated for early-stage squamous cell vulvar carcinoma local recurrence is reported in up to 40% after ten years. Knowledge on prognostic factors related to local recurrences should be helpful to select high risk patients and/or to develop strategies to prevent local recurrences. OBJECTIVE: This systematic review aims to evaluate the current knowledge on the incidence of local recurrences in vulvar carcinoma related to clinicopathologic and cell biologic variables. DATA SOURCES: Relevant studies were identified by an extensive online electronic search in July 2017. STUDY ELIGIBILITY CRITERIA: Studies reporting prognostic factors specific for local recurrences of vulvar carcinoma were included. STUDY APPRAISAL AND SYNTHESIS METHODS: Two review authors independently performed data selection, extraction and assessment of study quality. The risk difference was calculated for each prognostic factor when described in two or more studies. RESULTS: Twenty-two studies were included; most of all were retrospective and mainly reported pathologic prognostic factors. Our review indicates an estimated annual local recurrence rate of 4% without plateauing. The prognostic relevance for local recurrence of vulvar carcinoma of all analyzed variables remains equivocal, including pathologic tumor free margin distance <8mm, presence of lichen sclerosus, groin lymph node metastases and a variety of primary tumor characteristics (grade of differentiation, tumor size, tumor focality, depth of invasion, lymphovascular space invasion, tumor localization and presence of human papillomavirus). CONCLUSIONS: Current quality of data on prognostic factors for local recurrences in vulvar carcinoma patients does not allow evidence-based clinical decision making. Further research on prognostic factors, applying state of the art methodology is needed to identify high-risk patients and to develop alternative primary and secondary prevention strategies.

Quality of Life and Sexual Functioning After Vulvar Reconstruction With the Lotus Petal Flap.

OBJECTIVE: Resection of (pre) malignant lesions in the vulvoperineal area may result in large defects that cannot be closed primarily. The lotus petal flap technique is widely used for reconstruction. The aim of this study was to evaluate both quality of life (QoL) and sexual functioning of patients who underwent the lotus petal flap procedure, because no data are available on this topic. METHODS: A cross-sectional study was performed on all eligible patients (N = 38) who underwent the lotus petal flap procedure between 2005 and 2016. The European Organization for Research and Treatment of Cancer Quality of Life Questionnaire C30, Female Sexual Function Index, and Body Image Scale were used to evaluate QoL and sexual functioning. The European Organization for Research and Treatment of Cancer Quality of Life Questionnaire C30 and Female Sexual Function Index scores were compared with scores of age-matched healthy women. RESULTS: Twenty-six patients (68%) responded. The mean (SD) age was 65.5 (16.3) years, and the median follow-up time was 38.5 months (range 16-141 months). Quality of life scores were lower compared with healthy women in the domains physical, role, and social functioning. Sexual activity rates were comparable with healthy women; however, sexual functioning was worse. Although patients were satisfied about their sexual life, pain was reported. CONCLUSIONS: Patients who underwent vulvar reconstructive surgery with lotus petal flaps seem to have a lower QoL compared with healthy women. Patients report more pain during sexual activity but are satisfied about their sexual functioning. These results should be included in preoperative counseling and follow-up of future patients eligible for vulvar reconstruction with a lotus petal flap.

Surgical Treatment of Early-Stage Cervical Cancer: A Multi-Institution Experience in 2124 Cases in The Netherlands Over a 30-Year Period.

OBJECTIVE: This study aimed to describe the pattern of recurrence and survival related to prognostic variables, including type of surgery as a clinical variable, in patients surgically treated for early cervix cancer. METHODS: Records of 2124 patients who underwent a radical hysterectomy for International Federation of Gynaecology and Obstetrics stage I/IIA cervical cancer between 1982 and 2011 were reviewed. Clinical-pathologic prognostic variables, also including extent of parametrectomy, were identified and used in a multivariable Cox proportional hazard model to explore associations between disease-free survival (DFS) and prognostic variables. RESULTS: The 5-year DFS for the total group was 86%. Large tumor diameter, nonsquamous histology, lymph node metastases, parametrial involvement, lymph vascular space invasion, deep stromal invasion, and less radical surgery were independent poor prognostic variables for survival. Disease-free survival was independently associated with the type of radical hysterectomy with pelvic lymphadenectomy in favor of more radical parametrectomy (hazard ratio, 2.0; 95% confidence interval, 1.6-2.5). This difference was not found in tumors with a diameter of at least 20 mm. CONCLUSIONS: This study confirms that variables such as large tumor diameter, nonsquamous histology, lymph vascular space invasion, deep stromal invasion, positive lymph nodes, and parametrial infiltration are poor prognostic variables in early cervix cancer treated by surgery. The extent of parametrectomy had no influence on survival in tumors of 20 mm or less. For larger tumors, a more radical hysterectomy might be associated with better DFS. Taking into account the possible bias in this study as a result of its retrospective design, ideally a prospective cohort study with clear definition of radicality is necessary to answer this important clinical question.

Methylome analysis of extreme chemoresponsive patients identifies novel markers of platinum sensitivity in high-grade serous ovarian cancer.

BACKGROUND: Despite an early response to platinum-based chemotherapy in advanced stage high-grade serous ovarian cancer (HGSOC), the majority of patients will relapse with drug-resistant disease. Aberrant epigenetic alterations like DNA methylation are common in HGSOC. Differences in DNA methylation are associated with chemoresponse in these patients. The objective of this study was to identify and validate novel epigenetic markers of chemoresponse using genome-wide analysis of DNA methylation in extreme chemoresponsive HGSOC patients. METHODS: Genome-wide next-generation sequencing was performed on methylation-enriched tumor DNA of two HGSOC patient groups with residual disease, extreme responders (≥18 months progression-free survival (PFS), n = 8) and non-responders (≤6 months PFS, n = 10) to platinum-based chemotherapy. DNA methylation and expression data of the same patients were integrated to create a gene list. Genes were validated on an independent cohort of extreme responders (n = 21) and non-responders (n = 31) using pyrosequencing and qRT-PCR. In silico validation was performed using publicly available DNA methylation (n = 91) and expression (n = 208) datasets of unselected advanced stage HGSOC patients. Functional validation of FZD10 on chemosensitivity was carried out in ovarian cancer cell lines using siRNA-mediated silencing. RESULTS: Integrated genome-wide methylome and expression analysis identified 45 significantly differentially methylated and expressed genes between two chemoresponse groups. Four genes FZD10, FAM83A, MYO18B, and MKX were successfully validated in an external set of extreme chemoresponsive HGSOC patients. High FZD10 and MKX methylation were related with extreme responders and high FAM83A and MYO18B methylation with non-responders. In publicly available advanced stage HGSOC datasets, FZD10 and MKX methylation levels were associated with PFS. High FZD10 methylation was strongly associated with improved PFS in univariate analysis (hazard ratio (HR) = 0.43; 95% CI, 0.27-0.71; P = 0.001) and multivariate analysis (HR = 0.39; 95% CI, 0.23-0.65; P = 0.003). Consistently, low FZD10 expression was associated with improved PFS (HR = 1.36; 95% CI, 0.99-1.88; P = 0.058). FZD10 silencing caused significant sensitization towards cisplatin treatment in survival assays and apoptosis assays. CONCLUSIONS: By applying genome-wide integrated methylome analysis on extreme chemoresponsive HGSOC patients, we identified novel clinically relevant, epigenetically-regulated markers of platinum-sensitivity in HGSOC patients. The clinical potential of these markers in predictive and therapeutic approaches has to be further validated in prospective studies.

European Society of Gynaecological Oncology Guidelines for the Management of Patients With Vulvar Cancer.

OBJECTIVE: The aim of this study was to develop clinically relevant and evidence-based guidelines as part of European Society of Gynaecological Oncology's mission to improve the quality of care for women with gynecologic cancers across Europe. METHODS: The European Society of Gynaecological Oncology Council nominated an international development group made of practicing clinicians who provide care to patients with vulvar cancer and have demonstrated leadership and interest in the management of patients with vulvar cancer (18 experts across Europe). To ensure that the statements are evidence based, the current literature identified from a systematic search has been reviewed and critically appraised. In the absence of any clear scientific evidence, judgment was based on the professional experience and consensus of the development group (expert agreement). The guidelines are thus based on the best available evidence and expert agreement. Prior to publication, the guidelines were reviewed by 181 international reviewers including patient representatives independent from the development group. RESULTS: The guidelines cover diagnosis and referral, preoperative investigations, surgical management (local treatment, groin treatment including sentinel lymph node procedure, reconstructive surgery), radiation therapy, chemoradiation, systemic treatment, treatment of recurrent disease (vulvar recurrence, groin recurrence, distant metastases), and follow-up.

Re-expression of Selected Epigenetically Silenced Candidate Tumor Suppressor Genes in Cervical Cancer by TET2-directed Demethylation.

DNA hypermethylation is extensively explored as therapeutic target for gene expression modulation in cancer. Here, we re-activated hypermethylated candidate tumor suppressor genes (TSGs) (C13ORF18, CCNA1, TFPI2, and Maspin) by TET2-induced demethylation in cervical cancer cell lines. To redirect TET2 to hypermethylated TSGs, we engineered zinc finger proteins (ZFPs), which were first fused to the transcriptional activator VP64 to validate effective gene re-expression and confirm TSG function. ChIP-Seq not only revealed enriched binding of ZFPs to their intended sequence, but also considerable off-target binding, especially at promoter regions. Nevertheless, results obtained by targeted re-expression using ZFP-VP64 constructs were in line with cDNA overexpression; both revealed strong growth inhibition for C13ORF18 and TFPI2, but not for CCNA1 and Maspin. To explore effectivity of locus-targeted demethylation, ZFP-TET2 fusions were constructed which efficiently demethylated genes with subsequent gene re-activation. Moreover, targeting TET2 to TFPI2 and C13ORF18, but not CCNA1, significantly decreased cell growth, viability, and colony formation in cervical cancer cells compared to a catalytically inactive mutant of TET2. These data underline that effective re-activation of hypermethylated genes can be achieved through targeted DNA demethylation by TET2, which can assist in realizing sustained re-expression of genes of interest.

Studying platinum sensitivity and resistance in high-grade serous ovarian cancer: Different models for different questions.

High-grade serous ovarian cancer (HGSOC) has the highest mortality rate among all gynecological cancers. Patients are generally diagnosed in an advanced stage with the majority of cases displaying platinum resistant relapses. Recent genomic interrogation of large numbers of HGSOC patient samples indicated high complexity in terms of genetic aberrations, intra- and intertumor heterogeneity and underscored their lack of targetable oncogenic mutations. Sub-classifications of HGSOC based on expression profiles, termed 'differentiated', 'immunoreactive', 'mesenchymal' and 'proliferative', were shown to have prognostic value. In addition, in almost half of all HGSOC patients, a deficiency in homologous recombination (HR) was found that potentially can be targeted using PARP inhibitors. Developing precision medicine requires advanced experimental models. In the current review, we discuss experimental HGSOC models in which resistance to platinum therapy and the use of novel therapeutics can be carefully studied. Panels of better-defined primary cell lines need to be established to capture the full spectrum of HGSOC subtypes. Further refinement of cell lines is obtained with a 3-dimensional culture model mimicking the tumor microenvironment. Alternatively, ex vivo ovarian tumor tissue slices are used. For in vivo studies, larger panels of ovarian cancer patient-derived xenografts (PDXs) are being established, encompassing all expression subtypes. Ovarian cancer PDXs grossly retain tumor heterogeneity and clinical response to platinum therapy is preserved. PDXs are currently used in drug screens and as avatars for patient response. The role of the immune system in tumor responses can be assessed using humanized PDXs and immunocompetent genetically engineered mouse models. Dynamic tracking of genetic alterations in PDXs as well as patients during treatment and after relapse is feasible by sequencing circulating cell-free tumor DNA and analyzing circulating tumor cells. We discuss how various models and methods can be combined to delineate the molecular mechanisms underlying platinum resistance and to select HGSOC patients other than BRCA1/2-mutation carriers that could potentially benefit from the synthetic lethality of PARP inhibitors. This integrated approach is a first step to improve therapy outcomes in specific subgroups of HGSOC patients.

Identification of Analytical Factors Affecting Complex Proteomics Profiles Acquired in a Factorial Design Study with Analysis of Variance: Simultaneous Component Analysis.

Complex shotgun proteomics peptide profiles obtained in quantitative differential protein expression studies, such as in biomarker discovery, may be affected by multiple experimental factors. These preanalytical factors may affect the measured protein abundances which in turn influence the outcome of the associated statistical analysis and validation. It is therefore important to determine which factors influence the abundance of peptides in a complex proteomics experiment and to identify those peptides that are most influenced by these factors. In the current study we analyzed depleted human serum samples to evaluate experimental factors that may influence the resulting peptide profile such as the residence time in the autosampler at 4 °C, stopping or not stopping the trypsin digestion with acid, the type of blood collection tube, different hemolysis levels, differences in clotting times, the number of freeze-thaw cycles, and different trypsin/protein ratios. To this end we used a two-level fractional factorial design of resolution IV (2(IV)(7-3)). The design required analysis of 16 samples in which the main effects were not confounded by two-factor interactions. Data preprocessing using the Threshold Avoiding Proteomics Pipeline (Suits, F.; Hoekman, B.; Rosenling, T.; Bischoff, R.; Horvatovich, P. Anal. Chem. 2011, 83, 7786-7794, ref 1) produced a data-matrix containing quantitative information on 2,559 peaks. The intensity of the peaks was log-transformed, and peaks having intensities of a low t-test significance (p-value > 0.05) and a low absolute fold ratio (<2) between the two levels of each factor were removed. The remaining peaks were subjected to analysis of variance (ANOVA)-simultaneous component analysis (ASCA). Permutation tests were used to identify which of the preanalytical factors influenced the abundance of the measured peptides most significantly. The most important preanalytical factors affecting peptide intensity were (1) the hemolysis level, (2) stopping trypsin digestion with acid, and (3) the trypsin/protein ratio. This provides guidelines for the experimentalist to keep the ratio of trypsin/protein constant and to control the trypsin reaction by stopping it with acid at an accurately set pH. The hemolysis level cannot be controlled tightly as it depends on the status of a patient's blood (e.g., red blood cells are more fragile in patients undergoing chemotherapy) and the care with which blood was sampled (e.g., by avoiding shear stress). However, its level can be determined with a simple UV spectrophotometric measurement and samples with extreme levels or the peaks affected by hemolysis can be discarded from further analysis. The loadings of the ASCA model led to peptide peaks that were most affected by a given factor, for example, to hemoglobin-derived peptides in the case of the hemolysis level. Peak intensity differences for these peptides were assessed by means of extracted ion chromatograms confirming the results of the ASCA model.

Repeat sentinel lymph node procedure in patients with recurrent vulvar squamous cell carcinoma is feasible.

OBJECTIVE: Standard treatment of primary T1 squamous cell carcinoma (SCC) of the vulva <4cm consists of wide local excision (WLE) and sentinel lymph node (SLN) procedure of the groin(s). In case of a local recurrence WLE and inguino femoral lymphadenectomy (IFL) is generally recommended. In this study we assessed the feasibility of repeat SLN procedure in patients with recurrent vulvar SCC who were not able or willing to undergo IFL. METHODS: A retrospective study was performed in consecutive patients with recurrent vulvar SCC who underwent a repeat SLN procedure between 2006 and 2014. We present the clinical and pathological outcomes. The study conforms to the STROBE guidelines. RESULTS: A total number of 27 patients aged 35-87years at first diagnosis of SCC of the vulva were identified. Median follow-up after 2nd surgery was 27.4 (range 2-96) months. In 78% of patients and in 84% of the groins the repeat SLN procedure was successful. No structured questionnaires were used to describe details on the repeat SLN procedures but in general the gynecologic oncologists experienced repeat SLN procedures more challenging compared to primary procedures. There were no groin recurrences documented. CONCLUSIONS: Our findings suggest that it is feasible to perform a repeat SLN procedure in recurrent vulvar SCC, but the procedure appears technically more challenging compared to primary setting, resulting in a lower SLN identification rate.

European Network of Gynaecological Oncological Trial Groups' Requirements for Trials Between Academic Groups and Industry Partners--First Update 2015.

The first version of ENGOT's Requirements for Trials Between Academic Groups and Industry Partners in Europe was published 2010. This first update integrates the experiences made by the ENGOT network and the cooperative group studies while performing, analyzing, and publishing -among others - three large phase III trials. Furthermore, progress in European legislation and its impact on clinical studies in Europe have been considered in this update process.

Clinical validation of the Cervista HPV HR test according to the international guidelines for human papillomavirus test requirements for cervical cancer screening.

This study demonstrates that both the clinical sensitivity and specificity of the Cervista HPV HR test for high-risk human papillomavirus (HPV) detection are not inferior to those of the Hybrid Capture 2 (HC2) test. The intra- and interlaboratory reproducibilities of Cervista were 92.0% (kappa, 0.83) and 90.4% (kappa, 0.80), respectively. The Cervista HPV HR test fulfills all the international HPV test requirements for cervical primary screening purposes.

Radical surgery in patients with residual disease after (chemo)radiation for cervical cancer.

OBJECTIVE: The aim of this study was to determine possible impact of routinely scheduled biopsies and more radical surgery for residual central disease in locally advanced cervical cancer after (chemo)radiation. METHODS/MATERIALS: Data were analyzed of a consecutive series of cervical cancer patients (The International Federation of Gynecology and Obstetrics stages IB1-IVA) treated with (chemo) radiation between 1994 and 2011. Patients underwent gynecologic examination with biopsies 8 to 10 weeks after treatment. Since 2001, larger biopsies by electric loop excision were taken, and more radical surgery (type III hysterectomy or exenteration) was performed for central residual disease. Primary outcome was locoregional recurrence. Secondary outcomes were treatment-associated morbidity and disease-specific survival. RESULTS: Primary (chemo)radiation was given to 491 cervical cancer patients; 345 patients had a posttreatment biopsy. Viable tumor cells were identified in 84 patients, and 61 patients were eligible for salvage surgery. Residual disease after (chemo)radiation was an independent poor prognostic factor (hazard ratio, 3.59; 95% confidence interval, 2.18-5.93; P < 0.001). After 2001, larger biopsies were more frequently taken (29% vs 76%, P < 0.001), and in patients without viable tumor cells, locoregional recurrence after 2001 decreased from 21% to 10% (P = 0.01). After 2001, more patients underwent more radical surgery (46% vs 90%) (P < 0.001). Locoregional recurrence after surgery before 2001 occurred in 6 (46%) of the 13 patients, comparable with 19 (40%) of the 48 (P = 0.67) after 2001. More radical surgery was not associated with improved disease-specific survival (HR, 0.84; 95% CI, 0.20-3.46; P = 0.81) but did result in significantly more severe morbidity. CONCLUSION: More radical surgery in patients with (minimal) central residual disease identified by routine biopsy 8 to 10 weeks after (chemo)radiation does not improve survival and should not be recommended.

CADM1 and MAL promoter methylation levels in hrHPV-positive cervical scrapes increase proportional to degree and duration of underlying cervical disease.

Combined detection of cell adhesion molecule 1 (CADM1) and T-lymphocyte maturation-associated protein (MAL) promoter methylation in cervical scrapes is a promising triage strategy for high-risk human papillomavirus (hrHPV)-positive women. Here, CADM1 and MAL DNA methylation levels were analysed in cervical scrapes of hrHPV-positive women with no underlying high-grade disease, high-grade cervical intraepithelial neoplasia (CIN) and cervical cancer. CADM1 and MAL methylation levels in scrapes were first related to CIN-grade of the corresponding biopsy and second to CIN-grade stratified by the presence of 'normal' or 'abnormal' cytology as present in the accompanying scrape preceding the cervical biopsy. The scrapes included 167 women with ≤ CIN1, 54 with CIN2/3 and 44 with carcinoma. In a separate series of hrHPV-positive scrapes of women with CIN2/3 (n = 48), methylation levels were related to duration of preceding hrHPV infection (PHI; <5 and ≥ 5 years). Methylation levels were determined by quantitative methylation-specific PCR and normal cytology scrapes of hrHPV-positive women with histologically ≤ CIN1 served as reference. CADM1 and MAL methylation levels increased proportional to severity of the underlying lesion, showing an increase of 5.3- and 6.2-fold in CIN2/3, respectively, and 143.5- and 454.9-fold in carcinomas, respectively, compared to the reference. Methylation levels were also elevated in CIN2/3 with a longer duration of PHI (i.e. 11.5- and 13.6-fold, respectively). Moreover, per histological category, methylation levels were higher in accompanying scrapes with abnormal cytology than in scrapes with normal cytology. Concluding, CADM1 and MAL promoter methylation levels in hrHPV-positive cervical scrapes are related to the degree and duration of underlying cervical disease and markedly increased in cervical cancer.

The prevalence of underlying bleeding disorders in patients with heavy menstrual bleeding with and without gynecologic abnormalities.

OBJECTIVE: The purpose of this study was to assess the prevalence of underlying bleeding disorders in women with heavy menstrual bleeding (HMB) with and without gynecologic abnormalities. STUDY DESIGN: We performed a single-center prospective cohort study of 112 consecutive patients who were referred for heavy menstrual bleeding. Control subjects were 28 healthy volunteers who reported no HMB. Patients and control subjects had hemostatic testing in the first week after menstruation. Patients underwent gynecologic evaluation. RESULTS: The median age was 42.5 years (range, 17-55 years) in patients and 40.0 years (range, 25-55 years) in control subjects. Forty-six percent of patients had anemia; the median pictorial bleeding assessment chart score was 271. Seven percent of the control subjects with a subjectively normal menstruation had anemia. Twenty-six percent of patients had gynecologic abnormalities, which was considered to explain HMB. Overall, we found an underlying bleeding disorder in 29% of the patients, which was comparable for unexplained and explained HMB (31% vs 27%; P = .75). We diagnosed 6 cases of Von Willebrand's disease, 4 cases of factor XI deficiency, and 1 case of factor VII deficiency. The only abnormalities that we found in control subjects were platelet aggregation defects (11% in control subjects vs 23% in patients). Patients had a significantly longer activated partial thromboplastin time compared with control subjects (26.5 vs 25.0 seconds; P = .001) that was caused by lower median levels of factor XI (100 vs 124 IU/dL; P < .001). CONCLUSION: Bleeding disorders play an equally important role in the cause of both unexplained and explained heavy menstrual bleeding. A novel finding is the occurrence of low, but not deficient, levels of factor XI.

Simultaneous serum desalting and total protein determination by macroporous reversed-phase chromatography.

Macroporous reversed-phase (mRP) chromatography was successfully used to develop an accurate and precise method for total protein in serum. The limits of detection (0.83 µg, LOD) and quantification (2.51 µg, LOQ) for the mRP method are comparable with those of the widely used micro BCA protein assay. The mRP method can be used to determine the total protein concentration across a wide dynamic range by detecting chromatographic peaks at 215 nm and 280 nm. The method has the added advantage of desalting and denaturing proteins, leading to more complete digestion by trypsin and to better LC-MS-MS identification in shotgun proteomics experiments.

Multidimensional separation of tryptic peptides from human serum proteins using reversed-phase, strong cation exchange, weak anion exchange, and fused-core fluorinated stationary phases.

Proteome profiling of crude serum is a challenging task due to the wide dynamic range of protein concentrations and the presence of high-abundance proteins, which cover >90% of the total protein mass in serum. Peptide fractionation on strong cation exchange, weak anion exchange in the electrostatic repulsion hydrophilic interaction chromatography (ERLIC) mode, RP C18 at pH 2.5 (low pH), fused-core fluorinated at pH 2.5, and RP C18 at pH 9.7 (high pH) stationary phases resulted in two to three times more identified proteins and three to four times more identified peptides in comparison with 1D nanoChip-LC-MS/MS quadrupole TOF analysis (45 proteins, 185 peptides). The largest number of peptides and proteins was identified after prefractionation in the ERLIC mode due to the more uniform distribution of peptides among the collected fractions and on the RP column at high pH due to the high efficiency of RP separations and the complementary selectivity of both techniques to low-pH RP chromatography. A 3D separation scheme combining ERLIC, high-pH RP, and low-pH nanoChip-LC-MS/MS for crude serum proteome profiling resulted in the identification of 208 proteins and 1088 peptides with the lowest reported concentration of 11 ng/mL for heat shock protein 74.

Human papilloma virus 16 E6 RNA interference enhances cisplatin and death receptor-mediated apoptosis in human cervical carcinoma cells.

In cervical cancer, the p53 and retinoblastoma (pRb) tumor suppressor pathways are disrupted by the human papilloma virus (HPV) E6 and E7 oncoproteins, because E6 targets p53 and E7 targets pRb for rapid proteasome-mediated degradation. We have investigated whether E6 suppression with small interfering RNA (siRNA) restores p53 functionality and sensitizes the HPV16-positive cervical cancer cell line SiHa to apoptosis by cisplatin, irradiation, recombinant human tumor necrosis factor-related apoptosis-inducing ligand (rhTRAIL), or agonistic anti-Fas antibody. E6 siRNA resulted in decreased E6 mRNA levels and enhanced p53 and p21 expression, demonstrating the restoration of p53 functionality in SiHa cells, without inducing high levels of apoptosis (<10%). Cell surface expression of the proapoptotic death receptors (DRs) DR4, DR5, and Fas was not affected by E6 suppression. E6 suppression conferred susceptibility to cisplatin-induced apoptosis but not to irradiation-, rhTRAIL-, or anti-Fas antibody-induced apoptosis. Combining cisplatin with rhTRAIL or anti-Fas antibody induced even higher apoptosis levels in E6-suppressed cells. At the molecular level, cisplatin treatment resulted in elevated p53 levels, enhanced caspase-3 activation, and reduced p21 levels in E6-suppressed cells. Cisplatin in combination with death receptor ligands enhanced caspase-8 and caspase-3 activation and reduced X-linked inhibitor-of-apoptosis protein (XIAP) levels in these cells. We showed using siRNA that the enhanced apoptosis in E6-supressed cells was related to reduced XIAP levels and not due to reduced p21 levels. In conclusion, targeting E6 or XIAP in combination with cisplatin can efficiently potentiate rhTRAIL-induced apoptosis in HPV-positive cervical cancer cells.