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ObjectivesLow-grade serous ovarian carcinoma (LGSOC) is a distinct, rare, ovarian cancer type characterised by younger patient age and intrinsic chemoresistance. Understanding the molecular landscape is crucial for optimising targeted therapy.MethodsGenomic data from whole exome sequencing of tumour tissue was analysed in a LGSOC cohort with detailed clinical annotation.Results63 cases were analysed and three subgroups identified based on single nucleotide variants: canonical MAPK mutant (cMAPKm: 52%, KRAS/BRAF/NRAS), MAPK-associated gene mutation (MAPK-assoc: 27%) and MAPK wild-type (MAPKwt: 21%). NOTCH pathway disruption occurred across all subgroups. Tumour mutational burden (TMB), mutational signatures and recurrent copy number (CN) changes varied across the cohort with co-occurrence of chromosome 1p loss and 1q gain (CN Chr1pq) a recurrent feature. Low TMB and CN Chr1pq were associated with inferior disease-specific survival (HR 6.43; p < 0.001 and HR 3.29, p = 0.011 respectively). Stepwise genomic classification in relation to outcome resulted in four groups (TMB low; CN Chr1pq; MAPKwt/MAPKassoc; cMAPKm). 5 year disease-specific survival was 46%, 55%, 79% and 100% respectively for these groups. The two most favourable genomic subgroups were enriched for the SBS10b mutational signature, particularly the cMAPKm subgroup.ConclusionsLGSOC comprises multiple genomic subgroups with distinct clinical and molecular features. Chr1pq CN arm disruption and TMB represent promising methods to identify individuals with poorer prognosis. Further investigation of the molecular basis for these observations is required. MAPKwt cases represent around a fifth of patients. NOTCH inhibitors represent a candidate therapeutic strategy worthy of exploration across these cases.

Endometrial hyperplasia (EH) is a precursor lesion to endometrial carcinoma (EC). Risks for EC include genetic, hormonal and metabolic factors most notably those associated with obesity: rates are rising and there is concern that cases in pre-menopausal women may remain undetected. Making an accurate distinction between benign and pre-malignant disease is both a challenge for the pathologist and important to the gynaecologist who wants to deliver the most appropriate care to meet the needs of the patient. Premalignant change may be recognised by histological changes of endometrial hyperplasia (which may occur with or without atypia) and endometrial intraepithelial neoplasia (EIN).In this study we created a tissue resource of EH samples diagnosed between 2004 and 2009 (n=125) and used this to address key questions: 1. Are the EIN/WHO2014 diagnostic criteria able to consistently identify premalignant endometrium? 2. Can computer aided image analysis inform identification of EIN? 3. Can we improve diagnosis by incorporating analysis of protein expression using immunohistochemistry.Our findings confirmed the inclusion of EIN in diagnostic criteria resulted in a better agreement between expert pathologists compared with the previous WHO94 criteria used for the original diagnosis of our sample set. A computer model based on assessment of stromal:epithelial ratio appeared most accurate in classification of areas of tissue without EIN. From an extensive panel of putative endometrial protein tissue biomarkers a score based on assessment of HAND2, PTEN and PAX2 was able to identify four clusters one of which appeared to be more likely to be benign.In summary, our study has highlighted new opportunities to improve diagnosis of pre-malignant disease in endometrium and provide a platform for further research on this important topic

Background:\ud Low-grade serous carcinoma of the ovary or peritoneum is characterised by MAPK pathway aberrations and its reduced sensitivity to chemotherapy relative to high-grade serous carcinoma. We compared the MEK inhibitor trametinib to physician's choice standard of care in patients with recurrent low-grade serous carcinoma.\ud Methods:\ud This international, randomised, open-label, multicentre, phase 2/3 trial was done at 84 hospitals in the USA and UK. Eligible patients were aged 18 years or older with recurrent low-grade serous carcinoma and measurable disease, as defined by Response Evaluation Criteria In Solid Tumors version 1.1, had received at least one platinum-based regimen, but not all five standard-of-care drugs, and had received an unlimited number of previous regimens. Patients with serous borderline tumours or tumours containing low-grade serous and high-grade serous carcinoma were excluded. Eligible patients were randomly assigned (1:1) to receive either oral trametinib 2 mg once daily (trametinib group) or one of five standard-of-care treatment options (standard-of-care group): intravenous paclitaxel 80 mg/m2 by body surface area on days 1, 8, and 15 of every 28-day cycle; intravenous pegylated liposomal doxorubicin 40–50 mg/m2 by body surface area once every 4 weeks; intravenous topotecan 4 mg/m2 by body surface area on days 1, 8, and 15 of every 28-day cycle; oral letrozole 2·5 mg once daily; or oral tamoxifen 20 mg twice daily. Randomisation was stratified by geographical region (USA or UK), number of previous regimens (1, 2, or ≥3), performance status (0 or 1), and planned standard-of-care regimen. The primary endpoint was investigator-assessed progression-free survival while receiving randomised therapy, as assessed by imaging at baseline, once every 8 weeks for 15 months, and then once every 3 months thereafter, in the intention-to-treat population. Safety was assessed in patients who received at least one dose of study therapy. This trial is registered with ClinicalTrials.gov, NCT02101788, and is active but not recruiting.\ud Findings:\ud Between Feb 27, 2014, and April 10, 2018, 260 patients were enrolled and randomly assigned to the trametinib group (n=130) or the standard-of-care group (n=130). At the primary analysis, there were 217 progression-free survival events (101 [78%] in the trametinib group and 116 [89%] in the standard-of-care group). Median progression-free survival in the trametinib group was 13·0 months (95% CI 9·9–15·0) compared with 7·2 months (5·6–9·9) in the standard-of-care group (hazard ratio 0·48 [95% CI 0·36–0·64]; p

BACKGROUND: Low-grade serous carcinoma of the ovary or peritoneum is characterised by MAPK pathway aberrations and its reduced sensitivity to chemotherapy relative to high-grade serous carcinoma. We compared the MEK inhibitor trametinib to physician's choice standard of care in patients with recurrent low-grade serous carcinoma.METHODS: This international, randomised, open-label, multicentre, phase 2/3 trial was done at 84 hospitals in the USA and UK. Eligible patients were aged 18 years or older with recurrent low-grade serous carcinoma and measurable disease, as defined by Response Evaluation Criteria In Solid Tumors version 1.1, had received at least one platinum-based regimen, but not all five standard-of-care drugs, and had received an unlimited number of previous regimens. Patients with serous borderline tumours or tumours containing low-grade serous and high-grade serous carcinoma were excluded. Eligible patients were randomly assigned (1:1) to receive either oral trametinib 2 mg once daily (trametinib group) or one of five standard-of-care treatment options (standard-of-care group): intravenous paclitaxel 80 mg/m 2 by body surface area on days 1, 8, and 15 of every 28-day cycle; intravenous pegylated liposomal doxorubicin 40-50 mg/m 2 by body surface area once every 4 weeks; intravenous topotecan 4 mg/m 2 by body surface area on days 1, 8, and 15 of every 28-day cycle; oral letrozole 2·5 mg once daily; or oral tamoxifen 20 mg twice daily. Randomisation was stratified by geographical region (USA or UK), number of previous regimens (1, 2, or ≥3), performance status (0 or 1), and planned standard-of-care regimen. The primary endpoint was investigator-assessed progression-free survival while receiving randomised therapy, as assessed by imaging at baseline, once every 8 weeks for 15 months, and then once every 3 months thereafter, in the intention-to-treat population. Safety was assessed in patients who received at least one dose of study therapy. This trial is registered with ClinicalTrials.gov, NCT02101788, and is active but not recruiting. FINDINGS: Between Feb 27, 2014, and April 10, 2018, 260 patients were enrolled and randomly assigned to the trametinib group (n=130) or the standard-of-care group (n=130). At the primary analysis, there were 217 progression-free survival events (101 [78%] in the trametinib group and 116 [89%] in the standard-of-care group). Median progression-free survival in the trametinib group was 13·0 months (95% CI 9·9-15·0) compared with 7·2 months (5·6-9·9) in the standard-of-care group (hazard ratio 0·48 [95% CI 0·36-0·64]; pINTERPRETATION: Trametinib represents a new standard-of-care option for patients with recurrent low-grade serous carcinoma.FUNDING: NRG Oncology, Cancer Research UK, Target Ovarian Cancer, and Novartis.

More than 50% of all gynecologic tumors can be classified as rare (defined as an incidence of ≤6 per 100,000 women) and usually have a poor prognosis owing to delayed diagnosis and treatment. In contrast to almost all other common solid tumors, the treatment of rare gynecologic tumors (RGT) is often based on retrospective studies, expert opinion, or extrapolation from other tumor sites with similar histology, leading to difficulty in developing guidelines for clinical practice. Currently, gynecologic cancer research, due to distinct scientific and technological challenges, is lagging behind. Moreover, the overall efforts for addressing these challenges are fragmented across different European countries and indeed, worldwide. The GYNOCARE, COST Action CA18117 (European Network for Gynecological Rare Cancer Research) programme aims to address these challenges by creating a unique network between key stakeholders covering distinct domains from concept to cure: basic research on RGT, biobanking, bridging with industry, and setting up the legal and regulatory requirements for international innovative clinical trials. On this basis, members of this COST Action, (Working Group 1, “Basic and Translational Research on Rare Gynecological Cancer”) have decided to focus their future efforts on the development of new approaches to improve the diagnosis and treatment of RGT. Here, we provide a brief overview of the current state-of-the-art and describe the goals of this COST Action and its future challenges with the aim to stimulate discussion and promote synergy across scientists engaged in the fight against this rare cancer worldwide. publishersversion published

BACKGROUND: Approximately half of high-grade serous ovarian carcinomas (HGSOCs) demonstrate homologous recombination repair (HR) pathway defects, resulting in a distinct clinical phenotype comprising hypersensitivity to platinum, superior clinical outcome, and greater sensitivity to poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitors. EMSY, which is known to be amplified in breast and ovarian cancers, encodes a protein reported to bind and inactivate BRCA2. Thus, EMSY overexpression may mimic BRCA2 mutation, resulting in HR deficiency. However, to our knowledge, the phenotypic consequences of EMSY overexpression in HGSOC patients has not been explored.METHODS: Here we investigate the impact of EMSY expression on clinical outcome and sensitivity to platinum-based chemotherapy using available data from transcriptomically characterized HGSOC cohorts. RESULTS: High EMSY expression was associated with better clinical outcome in a cohort of 265 patients with HGSOC from Edinburgh (overall survival multivariable hazard ratio, 0.58 [95% CI, 0.38-0.88; P = .011] and progression-free survival multivariable hazard ratio, 0.62 [95% CI, 0.40-0.96; P = .030]). Superior outcome also was demonstrated in the Medical Research Council ICON7 clinical trial and multiple publicly available data sets. Patients within the Edinburgh cohort who had high EMSY expression were found to demonstrate greater rates of complete response to multiple platinum-containing chemotherapy regimens (radiological complete response rate of 44.4% vs 12.5% at second exposure; P = .035) and corresponding prolonged time to disease progression (median, 151.5 days vs 60.5 days after third platinum exposure; P = .004). CONCLUSIONS: Patients with HGSOCs demonstrating high EMSY expression appear to experience prolonged survival and greater platinum sensitivity, reminiscent of BRCA-mutant cases. These data are consistent with the notion that EMSY overexpression may render HGSOCs HR deficient.

BACKGROUND: Approximately half of high‐grade serous ovarian carcinomas (HGSOCs) demonstrate homologous recombination repair (HR) pathway defects, resulting in a distinct clinical phenotype comprising hypersensitivity to platinum, superior clinical outcome, and greater sensitivity to poly(adenosine diphosphate‐ribose) polymerase (PARP) inhibitors. EMSY, which is known to be amplified in breast and ovarian cancers, encodes a protein reported to bind and inactivate BRCA2. Thus, EMSY overexpression may mimic BRCA2 mutation, resulting in HR deficiency. However, to our knowledge, the phenotypic consequences of EMSY overexpression in HGSOC patients has not been explored.METHODS: Here we investigate the impact of EMSY expression on clinical outcome and sensitivity to platinum‐based chemotherapy using available data from transcriptomically characterized HGSOC cohorts.RESULTS: High EMSY expression was associated with better clinical outcome in a cohort of 265 patients with HGSOC from Edinburgh (overall survival multivariable hazard ratio, 0.58 [95% CI, 0.38‐0.88; P = .011] and progression‐free survival multivariable hazard ratio, 0.62 [95% CI, 0.40‐0.96; P = .030]). Superior outcome also was demonstrated in the Medical Research Council ICON7 clinical trial and multiple publicly available data sets. Patients within the Edinburgh cohort who had high EMSY expression were found to demonstrate greater rates of complete response to multiple platinum‐containing chemotherapy regimens (radiological complete response rate of 44.4% vs 12.5% at second exposure; P = .035) and corresponding prolonged time to disease progression (median, 151.5 days vs 60.5 days after third platinum exposure; P = .004).CONCLUSIONS: Patients with HGSOCs demonstrating high EMSY expression appear to experience prolonged survival and greater platinum sensitivity, reminiscent of BRCA‐mutant cases. These data are consistent with the notion that EMSY overexpression may render HGSOCs HR deficient.

The term ovarian cancer describes a heterogeneous group of tumours that grow in the ovary but are not necessarily of ovarian origin. Recent genomic analysis has shown that many of the most commonly used ovarian cancer cell lines have been mischaracterised, leading to erroneous conclusions and a gap in the translation of laboratory research into novel treatments for patients. Here, we use 10 epithelial ovarian cancer cell lines to investigate 2D migration, cell cycle parameters and 3D invasion behaviour into different substrates and find significant differences between the behaviours of cell lines from different origins. Cell lines derived from non-serous carcinomas migrated more quickly and were more likely to invade into Matrigel and collagen I substrates than cell lines derived from high-grade serous carcinomas. However not all cell lines derived from non-serous carcinomas exhibited similar invasive behaviour. These findings may reflect differences in the behaviour of the primary tumour types from which the cell lines were derived, given that high-grade serous carcinomas typically expand and spread over peritoneal surfaces. These results provide the basis of an in vitro model for identifying differences between ovarian cancer tumour types.

The aim of this article is to propose guidelines and recommendations in problematic areas in pathologic reporting of endometrial carcinoma (EC) regarding special techniques and ancillary studies. An organizing committee designed a comprehensive survey with different questions related to pathologic features, diagnosis, and prognosis of EC that was sent to all members of the International Society of Gynecological Pathologists. The special techniques/ancillary studies group received 4 different questions to be addressed. Five members of the group reviewed the literature and came up with recommendations and an accompanying text which were discussed and agreed upon by all members of the group. Twelve different recommendations are made. They address the value of immunohistochemistry, ploidy, and molecular analysis for assessing prognosis in EC, the value of steroid hormone receptor analysis to predict response to hormone therapy, and parameters regarding applying immunohistochemistry and molecular tests for assessing mismatch deficiency in EC.

This work was supported by the Engineering and Physical Sciences Research Council and Medical Research Council [EP/L016559/1, EP/P030017/1], and CRUK [A18075 Core Award]. The ability to provide quantitative, objective and automated pathological analysis would provide enormous benefits for national cancer screening programmes, in terms of both resource reduction and improved patient wellbeing. The move towards molecular pathology through spectroscopic methods shows great promise, but has been restricted by spectral quality, acquisition times and lack of direct clinical application. In this paper, we present the application of wavelength modulated Raman spectroscopy for the automated label- and fluorescence-free classification of fixed squamous epithelial cells in suspension, such as those produced during a cervical smear test. Direct comparison with standard Raman spectroscopy shows marked improvement of sensitivity and specificity when considering both human papillomavirus (sensitivity +12.0%, specificity +5.3%) and transformation status (sensitivity +10.3%, specificity +11.1%). Studies on the impact of intracellular sampling location and storage effects suggest that wavelength modulated Raman spectroscopy is sufficiently robust to be used in fixed cell classification, but requires further investigations of potential sources of molecular variation in order to improve current clinical tools. Publisher PDF

Globally, two billion people suffer from micronutrient malnutrition. Biofortification, the process of breeding staple food crops to have higher micronutrient content, has proven to be efficacious and cost-effective in addressing micronutrient malnutrition. To determine where and in which crop-micronutrient combinations to invest, a global Biofortification Prioritization Index (BPI) was developed (Asare-Marfo et al., 2013). While a country s rank in the global context is useful, it is not granular enough to develop strategies within heterogenous countries. Therefore, this paper utilizes methodology to develop a subnational-level BPI for Nigeria, a country which shows promise for biofortified crops. The subnational BPI is based on three sub-indices: production, consumption, and micronutrient deficiency. In addition, targeted areas are classified as areas of: (1) impact and intervention, (2) impact, or (3) intervention. Sensitivity analyses tested the robustness of BPI results on single sub-index parameters. For vitamin A maize s introduction, the North East and North West zones offer the most promise while the southern zones generate the greatest impact for the introduction of vitamin A cassava. Concentrating vitamin A sweet potato investments in the North Central zone is the most effective while focusing in the North West is the most promising strategy for iron pearl millet. Acknowledgement : The authors would sincerely like to thank Dr. Erick Boy, Head of the HarvestPlus Nutrition Research Unit, and Ms. Amarachi Utah for their consultation and support of this research.

Focusing ultrashort laser pulses in time in tandem with single-pixel detection images at depth with no sample characterization., Optical approaches to fluorescent, spectroscopic, and morphological imaging have made exceptional advances in the last decade. Super-resolution imaging and wide-field multiphoton imaging are now underpinning major advances across the biomedical sciences. While the advances have been startling, the key unmet challenge to date in all forms of optical imaging is to penetrate deeper. A number of schemes implement aberration correction or the use of complex photonics to address this need. In contrast, we approach this challenge by implementing a scheme that requires no a priori information about the medium nor its properties. Exploiting temporal focusing and single-pixel detection in our innovative scheme, we obtain wide-field two-photon images through various turbid media including a scattering phantom and tissue reaching a depth of up to seven scattering mean free path lengths. Our results show that it competes favorably with standard point-scanning two-photon imaging, with up to a fivefold improvement in signal-to-background ratio while showing significantly lower photobleaching.

Normal development of the uterine cervix has been widely studied and the origin of both the columnar and squamous epithelia, as well as the molecular basis of their differentiation, has been established. The process of early carcinogenesis in the uterine cervix has also been described extensively, in particular with respect to the role of human papillomavirus (HPV) infection. However, questions remain about the progenitor cell(s) that play(s) a role in normal (embryonic and fetal) development, as well as in the oncogenic processes that take place in the transformation zone of the uterine cervix. This chapter describes the development of the human lower female reproductive tract, in particular the cervical squamocolumnar junction, and its implications for the pathogenesis of cervical cancer.

Source: https://erdc-library.erdc.dren.mil/jspui/ An undistorted scale hydraulic model study was conducted to investigate the stability response of modified repair options for the San Pedro Breakwater. A plan which employs 13- to 20-ton armor stone was developed. This plan proved to be more stable than the original breakwater section for both 45- and 90-deg wave attack.

Source: https://erdc-library.erdc.dren.mil/jspui/ The purpose of the investigation herein was to obtain design information for stone and dolos armor used on breakwater heads and subjected to nonbreaking waves. More specifically, it was desired to determine the minimum weight of individual armor units (with given specific weights) required for stability as a function of: (a.) Type of armor unit. (b.) Sea-side slope of the structure. (c.) Angle of wave attack. (d.) Wave period. (e.) Wave height. Based on tests and results described herein, in which stone and dolos armor are used on conical breakwater heads and subjected to nonbreaking waves with angles of wave attack of 0, 45, 90, and 135 deg, it is concluded that: (a.) The longer wave periods (2.00 and 2.75 sec) generally produce the lower stabilities. (b.) Angles of wave attack of 45 and 90 deg are the most critical. (c.) Flattening the slope from 1V on 1.5H to 1V on 2H does not improve stability of the stone armor.

Source: https://erdc-library.erdc.dren.mil/jspui/ A Hydraulic Model Investigation was conducted at geometrically undistorted, linear scales of 1:31 and 1:25, model to prototype, to evaluate the stability against wave attack of three areas of the existing breakwater at Nawiliwili Harbor, Kauai, Hawaii. Where the existing crown and/or harbor-side slope proved to be unstable, additional tests were conducted to check the stability of the rehabilitation designs proposed by the Pacific Ocean Division. All existing and proposed rehabilitation designs were tested for the worst breaking wave conditions that could be produced for the selected wave periods, water depths, and bathymetry seaward of the test sections. The existing, 22,000-lb dolosse on the sea-side slope at sta 19+50 proved to be unstable for the wave conditions of Hydrograph I (maximum wave height of 24.5 ft), while the remainder of the breakwater cross section (concrete crown cap and 16,000- to 20,000-lb stone on harbor-side slope) proved to be stable for the selected test conditions. The stability of the existing breakwater cross section at sta 14+00 proved to be dependent upon how tight a keyed and fitted construction of the 16,000- to 20,000-lb armor stone actually exists on the crown and harbor-side slope. Model tests results using Hydrograph II (maximum wave height of 22.5 ft) indicated that if a tight, keyed and fitted construction exists, the breakwater at sta 14+00 is an adequate design, but if this tight construction does not exist, the crown and harbor-side slope could sustain severe damage and this damage could result in the undermining and displacement of existing dolosse (22,000 lb) along the breakwater crown. A concrete rib cap and one layer of uniformly placed, 13,000-lb tribars proved to be adequate rehabilitation designs for the crown and harbor-side slope, respectively, at sta 14+00. The existing 14,000- to 24,000-lb random-placed armor stone on the sea-side slope, and 16,000- to 20,000-lb keyed and fitted armor stone on the crown and harbor-side slope at sta 10+00 proved to be adequate designs for t he selected test conditions of Hydrograph III (maximum wave height of 11.6 ft).

Contains fulltext : 283536.pdf (Publisher’s version ) (Closed access) The Gynecologic Cancer InterGroup (GCIG) sixth Ovarian Cancer Conference on Clinical Research was held virtually in October, 2021, following published consensus guidelines. The goal of the consensus meeting was to achieve harmonisation on the design elements of upcoming trials in ovarian cancer, to select important questions for future study, and to identify unmet needs. All 33 GCIG member groups participated in the development, refinement, and adoption of 20 statements within four topic groups on clinical research in ovarian cancer including first line treatment, recurrent disease, disease subgroups, and future trials. Unanimous consensus was obtained for 14 of 20 statements, with greater than 90% concordance in the remaining six statements. The high acceptance rate following active deliberation among the GCIG groups confirmed that a consensus process could be applied in a virtual setting. Together with detailed categorisation of unmet needs, these consensus statements will promote the harmonisation of international clinical research in ovarian cancer.

A key effect of human papillomavirus (HPV) infection is to disrupt the normal cell cycle in order to subvert the cellular DNA replication machinery. Morphologically, condylomata induced by high and low risk HPV types cannot be distinguished and many studies have shown that the pattern of viral gene expression is similar in condylomata caused by both high risk and low risk HPV types. Detailed morphological study of cell cycle protein expression has not previously been performed on condylomata infected with low risk HPV types. The findings presented suggest that the mechanisms employed by low risk HPV6 or HPV11 to subvert cellular functions in condylomata acuminata are similar to those employed by high risk HPVs, with the exception of cyclin D1 and p53 protein over-expression. The differences in p53 expression and cyclin D1 expression seen between high and low risk HPV infection, reflect the known differences between high and low risk types and are in agreement with the known differences between high risk and low risk E6 and E7 proteins. PHK transduction studies demonstrated HPV E6 and E7 induce changes in cell cycle protein expression and that there are differences in cell cycle abrogation between HPV6 and HPV16. Disruption of the p53-MDM2 interaction can lead to activation of the p53 pathway. HPV infected lesions almost always contain wild-type p53. The binding of HPV E6 to p53, and its subsequent targeting for degradation, prevents activation of the p53 pathway in HPV infected cells. Cells over expressing HPV genes were treated with Nutlin-3, a MDM2-small molecule antagonist. The findings presented suggest treatment with Nutlin-3 induces cell cycle arrest in cells expressing HPV16 E7 and HPV6 E6 and HPV6 E7. This suggests a potential role for Nutlin-3 in the treatment of HPV infected cells.

AIMS: To determine the relative diagnostic performance of non-isotopic in situ hybridisation (NISH) and a dot-blot assay for detecting human papillomavirus (HPV) on exfoliated cervical cells; and to correlate the results with cytopathological assessment. METHODS: Cervical smears and cytological samples were obtained from 122 patients during the same clinical examination and the presence of HPV sequences determined by NISH and dot-blot analysis, respectively. RESULTS: Dot-blot analysis gave an autoradiographic signal in 15 of 121 (12.4%) cases, while NISH detected viral genomes in 38 of 114 (33.3%) cases. Even in the presence of koilocytosis, where vegetative replication of the virus occurs, NISH was positive in over twice as many cases as dot-blot analysis (NISH 90%, dot-blot 40%), while in smears within normal cytological limits, where the viral copy number is likely to be considerably lower, the differences were more striking (NISH 31%, dot-blot 5%). CONCLUSIONS: These data show that NISH on cytological smears is more sensitive than a standardised dot-blot hybridisation assay for detecting HPV infection in cytological material and is therefore a more appropriate screening tool.

Cervical cancer is a major cause of death in women and is strongly associated with infection by human papillomavirus (HPV). Integration of HPV is thought to form a key step in the formation of cancer, and is thought to involve the upregulation of HPV E6 and E7 due to the loss of E2 transcriptional control. Leptomycin B (LMB), a nuclear export inhibitor, has previously been shown to induce apoptosis in HPV-containing cancer cell lines and HPV 16 E7 or E6/E7 transduced primary keratinocytes, but not in normal cells. This thesis shows that LMB can induce apoptosis and a reduction in the colony survival of derivatives of the W12 cell line that contain HPV 16 in either episomal or integrated form. The HPV genome status, including variations in viral integration type, appears to influence the cumulative and temporal pattern of LMB-induced apoptosis. The effects of LMB were also apparent in cells grown in organotypic raft culture, with differences in behaviour again apparent between cells containing episomal and integrated HPV. As previously noted, treatment with LMB was associated with increased expression of the cell regulators p53 and p21; however, the induction of apoptosis was not dependent upon transcriptionally active p53. It is therefore likely that induction and mediation of LMB-induced apoptosis occurs via alternative, currently unidentified, pathways. These findings suggest that LMB can induce apoptosis in keratinocytes containing HPV 16 in either episomal or integrated form, with genome status and potentially lesion grade likely to influence the response of HPV-associated anogenital lesions to LMB treatment.

AIMS--To assess the relative diagnostic performance of the polymerase chain reaction (PCR) and non-isotopic in situ hybridisation (NISH) and to correlate these data with cytopathological assessment. METHODS--Paired analysis of human papillomavirus (HPV) detection was performed by PCR and NISH on exfoliated cervical cells from 122 women attending a routine gynaecological examination. PCR amplification followed by generic and HPV type specific hybridisation was compared with NISH on a parallel cervical smear. RESULTS--Overall, 32 cases were positive by NISH and 61 positive by PCR. Of the 105 cases in which both PCR and NISH were interpretable, 76 (26%) were normal smears, 20 of which were HPV positive by NISH and 37 (49%) by PCR. Of 17 borderline smears, two were NISH positive and 12 PCR positive. Eight of nine smears containing koilocytes were positive by NISH and seven by PCR. Of three dyskaryotic smears, none were NISH and two were PCR positive. The concordance of NISH and PCR in these samples was 57%. To assess sampling error, NISH and PCR were performed on an additional 50 cases using aliquots from the same sample. This increased the concordance between assays to 74%. Filter hybridisation of PCR products with the cocktail of probes used in NISH (under low and high stringency conditions) demonstrated that several cases of NISH positivity could be accounted for by cross-hybridisation to HPV types identified by PCR but not present in the NISH probe cocktail. CONCLUSIONS--Sampling error and potential cross-hybridisation of probe and target should be considered in interpretation of these techniques. PCR is more sensitive because it provides for the amplification of target DNA sequences. In addition, the PCR assay utilised in this study detects a wider range of HPV types than are contained in the cocktails used for NISH. However, PCR assays detect viral DNA present both within cells and in cervical fluid whereas NISH permits morphological localisation.

Routine cervical smears (n = 262) from a Sexually Transmitted Diseases clinic were screened by non-isotopic in situ hybridization (NISH) stratifying human papillomavirus (HPV) infections into HPV6/11 (low risk) and HPV16/18/33 (high risk) categories. Of 188 patients with cytologically normal smears, HPV sequences were demonstrated in 41%. Of the 128 cases analysed by dual NISH, 16% contained low risk, 20% high risk and 5% both groups. In patients with cytological evidence of wart virus infection (WVI) only, 54% (n = 50) contained high-risk and 22% low-risk HPV types. The comparable incidences in CIN1/2 plus WVI (n = 24) were not significantly different: 54% and 17%, respectively. Cytological criteria underestimate the prevalence of HPV infection in patients with cytologically normal smears. This represents either 'occult' or 'latent' infection. The identical prevalence of HPVB16/18/33 in WVI only, and CIN1/2 plus WVI, suggests that the cytopathic effect induced by these HPVs may represent one end of a spectrum of morphological change which progresses to cervical intraepithelial neoplasia (CIN).

AIMS: To determine the relative diagnostic sensitivity of non-isotopic in situ hybridisation (NISH) for the diagnosis of human papillomavirus (HPV) on matched smears and biopsy specimens; to compare the NISH signal type in the two samples; and to correlate the NISH data with the morphological diagnosis. METHODS: HPV samples were assayed individually by NISH with digoxigenin labelled probes (HPV6, 11, 16, 18, and 33) on routinely collected paraffin wax embedded cervical biopsy specimens and for high risk HPVs with a cocktail of similarly labelled probes (HPV16, 18, 33) on matched smears. These were taken at the same colposcopic examination from 32 patients investigated for an abnormal cervical Papanicolaou (PAP) stained smear. RESULTS: An HPV signal was present in 18 (56%) biopsy specimens and in 14 (44%) smears. There was higher concordance of sets of data in the presence of cytopathic wart virus changes. The superiority of biopsy over smear in detecting HPV was mainly the result of examining the entire cervical biopsy specimen rather than cells scraped from the cervical surface. The NISH signal type in both biopsy specimen and smear was similar; it has been shown that NISH type 1 signal correlates with episomal viral replication and type 2 and 3 signals with viral integration. CONCLUSIONS: These data show that NISH on cervical smears is a worthwhile primary screen for HPV infection. The NISH signal types in cervical smears are similar to those previously described in cervical biopsy specimens.

AIM: To develop a methodology for direct detection of high risk human papillomavirus (HPV) infection in routine cervical smears by non-isotopic in situ hybridisation (NISH) which can be compared with cytopathological assessment of the same cells. METHODS: The methodology was established using cultured cells and routine cervical smears hybridised with digoxigenin labelled probes for HPV, 16, 18, 31, and 33. The technique was applied to the analysis of 53 patients from a sexually transmitted disease clinic. RESULTS: The optimal sensitivity achieved for single HPV detection in cultured cells was 1-2 copies of HPV 16 per cell and that for detection of a cocktail of HPV types in routine cervical smears was 2.5-12 copies per cell. Of parallel smears taken from patients with a normal Papinacolau-stained smear 33.3% (24) contained a HPV 16, 18, 31, and 33 signal indicating an occult HPV infection. The prevalence of these HPV types was similar in women in whom a cytopathological diagnosis of wart virus infection was made (64.7%, 17) and in patients with mild dyskaryosis (75%, 12). CONCLUSIONS: The methodology evolved localises HPV sequences directly to epithelial cell nuclei, which can be morphologically assessed by haematoxylin counterstaining. Sample contamination with exogenous viral sequences can be distinguished from true infection. In this study, a HPV signal was not found in morphologically normal epithelial cells. The methods described will permit the detection of HPV sequences in routinely collected cervical smears and the evaluation of the natural history and potential clinical relevance of HPV infection without changes in clinical practice.