Yin Ling Woo

A prospective study on the natural course of low-grade squamous intraepithelial lesions and the presence of HPV16 E2-, E6- and E7-specific T-cell responses.

This study investigates the clinical course of low grade squamous intraepithelial lesions (LSIL), HPV status and HPV16‐specific immune response in a large prospective study of 125 women with LSIL followed cytologically, virologically and histologically. Women with low‐grade abnormal smears were recruited and followed‐up for one year. Colposcopy, cervical biopsy for histology and brushings for HPV typing was performed at recruitment, 6 months (no biopsy) and upon completion of the study at one year. HPV16‐specific T‐cell responses were analysed by interferon‐γ ELISPOT at entry, 6 and 12 months. Infection with multiple HPV types was detected in 70% of all patients, HPV16 was found in 42% of the patients. LSIL lesions progressed to HSIL in 24%, persisted in 60% and regressed to normal in 16% of the patients. No difference was observed in the clearance rate of infections with single or multiple HPV types among the groups with a different histological outcome. HPV16‐specific type 1 T‐cell responses were detected in only half of the patients with an HPV16+ LSIL, and predominantly reactive to HPV16 E2 and E6. Interestingly, the presence of HPV16 E2‐specific T‐cell responses correlated with absence of progression of HPV16+ lesions (p = 0.005) while the detection of HPV16 E6 specific reactivity was associated with persistence (p = 0.05). This large prospective study showed that the majority of LSIL persisted or progressed within the first year.This was paralleled by immune failure as most of the patients with an HPV16+ LSIL failed to react to peptides of HPV16 E2, E6 or E7.

Centralisation of services for gynaecological cancers - a Cochrane systematic review.

OBJECTIVE Gynaecological cancers are the second most common cancers among women. It has been suggested that centralised care improves outcomes but consensus is lacking. This systematic review assesses the effectiveness of centralisation of care for patients with gynaecological cancer, in particular, survival advantage. METHODS A comprehensive search of the Cochrane Gynaecological Cancer Group Trials Register, CENTRAL (The Cochrane Library, Issue 4, 2010), MEDLINE, and EMBASE up to November 2010 was conducted. Registers of clinical trials, abstracts of scientific meetings, and reference lists of included studies were also searched. Randomised controlled trials (RCTs), quasi-RCTs, controlled before-and-after studies, interrupted time series studies, and observational studies were included and multivariable analysis to adjust for baseline case mix were used. RESULTS Five retrospective observational studies met the inclusion criteria. Meta-analysis of three studies assessing over 9000 women suggested that institutions with gynaecologic oncologists on site may prolong survival in women with ovarian cancer, compared to community or general hospitals: hazard ratio (HR) of death was 0.90 (95% confidence interval (CI) 0.82 to 0.99). Similarly, another meta-analysis of three studies assessing over 50,000 women, found that teaching centres or regional cancer centres may prolong survival in women with any gynaecological cancer compared to community or general hospitals (HR 0.91; 95% CI 0.84 to 0.99). The largest of these studies included all gynaecological malignancies and assessed 48,981 women, so the findings extend beyond ovarian cancer. One study compared community hospitals with semi-specialised gynaecologists versus general hospitals and reported non-significantly better disease-specific survival in women with ovarian cancer (HR 0.89; 95% CI 0.78 to 1.01). The findings of included studies were highly consistent. CONCLUSIONS The meta-analysis provides evidence to suggest that women with gynaecological cancer who received treatment in specialised centres had longer survival than those managed elsewhere.

Identification of Human Papillomavirus Type 58 Lineages and the Distribution Worldwide

BACKGROUND Human papillomavirus type 58 (HPV-58) accounts for a much higher proportion of cervical cancers in East Asia than other types. A classification system of HPV-58, which is essential for molecular epidemiological study, is lacking. METHODS AND RESULTS This study analyzed the sequences of 401 isolates collected from 15 countries and cities. The 268 unique concatenated E6-E7-E2-E5-L1-LCR sequences that comprised 57% of the whole HPV-58 genome showed 4 distinct clusters. L1 and LCR produced tree topologies that best resembled the concatenated sequences and thus are the most appropriate surrogate regions for lineage classification. Moreover, short fragments from L1 (nucleotides 6014-6539) and LCR (nucleotides 7257-7429 and 7540-52) were found to contain sequence signatures informative for lineage identification. Lineage A was the most prevalent lineage across all regions. Lineage C was more frequent in Africa than elsewhere, whereas lineage D was more prevalent in Africa than in Asia. Among lineage A variants, sublineage A2 dominated in Africa, the Americas, and Europe, but not in Asia. Sublineage A1, which represents the prototype that originated from a patient with cancer, was rare worldwide except in Asia. CONCLUSIONS HPV-58 can be classified into 4 lineages that show some degree of ethnogeographic predilection in distribution. The evolutionary, epidemiological, and pathological characteristics of these lineages warrant further study.

Characterising the local immune responses in cervical intraepithelial neoplasia: a cross-sectional and longitudinal analysis

Introduction  Immunological competence influences the progression of cervical intraepithelial neoplasia (CIN) to invasive cancer. Information on the local immunological changes during the natural course of CIN is central for the development of new therapies.

Cancer-Associated Fibroblasts Promote Proliferation of Endometrial Cancer Cells

Endometrial cancer is the most commonly diagnosed gynecologic malignancy worldwide; yet the tumor microenvironment, especially the fibroblast cells surrounding the cancer cells, is poorly understood. We established four primary cultures of fibroblasts from human endometrial cancer tissues (cancer-associated fibroblasts, CAFs) using antibody-conjugated magnetic bead isolation. These relatively homogenous fibroblast cultures expressed fibroblast markers (CD90, vimentin and alpha-smooth muscle actin) and hormonal (estrogen and progesterone) receptors. Conditioned media collected from CAFs induced a dose-dependent proliferation of both primary cultures and cell lines of endometrial cancer in vitro (175%) when compared to non-treated cells, in contrast to those from normal endometrial fibroblast cell line (51%) (P<0.0001). These effects were not observed in fibroblast culture derived from benign endometrial hyperplasia tissues, indicating the specificity of CAFs in affecting endometrial cancer cell proliferation. To determine the mechanism underlying the differential fibroblast effects, we compared the activation of PI3K/Akt and MAPK/Erk pathways in endometrial cancer cells following treatment with normal fibroblasts- and CAFs-conditioned media. Western blot analysis showed that the expression of both phosphorylated forms of Akt and Erk were significantly down-regulated in normal fibroblasts-treated cells, but were up-regulated/maintained in CAFs-treated cells. Treatment with specific inhibitors LY294002 and U0126 reversed the CAFs-mediated cell proliferation (P<0.0001), suggesting for a role of these pathways in modulating endometrial cancer cell proliferation. Rapamycin, which targets a downstream molecule in PI3K pathway (mTOR), also suppressed CAFs-induced cell proliferation by inducing apoptosis. Cytokine profiling analysis revealed that CAFs secrete higher levels of macrophage chemoattractant protein (MCP)-1, interleukin (IL)-6, IL-8, RANTES and vascular endothelial growth factor (VEGF) than normal fibroblasts. Our data suggests that in contrast to normal fibroblasts, CAFs may exhibit a pro-tumorigenic effect in the progression of endometrial cancer, and PI3K/Akt and MAPK/Erk signaling may represent critical regulators in how endometrial cancer cells respond to their microenvironment.

Geographical distribution and oncogenic risk association of human papillomavirus type 58 E6 and E7 sequence variations.

Human papillomavirus (HPV) 58 accounts for a notable proportion of cervical cancers in East Asia and parts of Latin America, but it is uncommon elsewhere. The reason for such ethnogeographical predilection is unknown. In our study, nucleotide sequences of E6 and E7 genes of 401 HPV58 isolates collected from 15 countries/cities across four continents were examined. Phylogenetic relationship, geographical distribution and risk association of nucleotide sequence variations were analyzed. We found that the E6 genes of HPV58 variants were more conserved than E7. Thus, E6 is a more appropriate target for type‐specific detection, whereas E7 is more appropriate for strain differentiation. The frequency of sequence variation varied geographically. Africa had significantly more isolates with E6‐367A (D86E) but significantly less isolates with E6‐203G, ‐245G, ‐367C (prototype‐like) than other regions (p ≤ 0.003). E7‐632T, ‐760A (T20I, G63S) was more frequently found in Asia, and E7‐793G (T74A) was more frequent in Africa (p < 0.001). Variants with T20I and G63S substitutions at E7 conferred a significantly higher risk for cervical intraepithelial neoplasia grade III and invasive cervical cancer compared to other HPV58 variants (odds ratio = 4.44, p = 0.007). In conclusion, T20I and/or G63S substitution(s) at E7 of HPV58 is/are associated with a higher risk for cervical neoplasia. These substitutions are more commonly found in Asia and the Americas, which may account for the higher disease attribution of HPV58 in these areas.

Sensitive HPV detection in oropharyngeal cancers

BackgroundHuman papillomaviruses (HPV) are the aetiological agents of certain benign and malignant tumours of skin and mucosae; the most important of which is cervical cancer. Also, the incidence of ano-genital warts, HPV-anal cancer and oropharyngeal cancers are rising. To help ascertain a useful PCR detection protocol for oropharyngeal cancers, we directly compared three commonly used primer sets in detection of HPV from different clinical samples.MethodsWe compared PGMY09/11, MY09/11 and GP5+/6+ primers sets in PCRs of 34 clinically diagnosed samples of genital warts, cervical brushings (with associated histological diagnosis) and vulval biopsies. All negative samples were subsequently tested using the previously reported PGMY/GP PCR method and amplicons directly sequenced for confirmation and typing. An optimised PCR protocol was then compared to a line blot assay for detection of HPV in 15 oropharyngeal cancer samples.ResultsPGMY09/11 primers detected HPV presence in more cervical brushing (100%) and genital wart (92.9%) samples compared to MY09/11 (90% and 64.3%) and GP5+/6+ (80% and 64.3%) primer sets, respectively. From vulval biopsies, HPV detection rates were: MY09/11 (63.6%), GP5+/6+ (54.5%) and PGMY09/11 (54.5%). PGMY/GP nested PCR demonstrated that HPV was present, and direct sequencing confirmed genotypes. This nested PCR protocol showed detection of HPV in 10/15 (66.7%) of oropharyngeal cancer samples.ConclusionsPGMY09/11 primers are the preferred primer set among these three for primary PCR screening with different clinical samples. MY09/11 and GP5+/6+ may be used (particularly for cervical samples) but demonstrate lower detection rates. A nested PCR approach (i.e. a PGMY-GP system) may be required to confirm negativity or to detect low levels of HPV, undetectable using current primary PCR methods, as demonstrated using oropharyngeal cancer samples.

Expression of neuronal markers in the endometrium of women with and those without endometriosis

STUDY QUESTION How do the expression patterns of neuronal markers differ in the endometrium of women with and without endometriosis? SUMMARY ANSWER The neuronal markers, PGP9.5, NGFp75 and VR1, are expressed in the endometrium at levels that do not differ between women with and without endometriosis. WHAT IS KNOWN ALREADY Aberrant neuronal growth within the uterus may contribute to abnormal fertility and uterine dysfunction. However, controversy still exists as to whether aberrant innervation in the endometrium is associated with gynaecological pathology such as endometriosis. This may reflect the use of subjective methods such as histology to assess the innervation of the endometrium. We, therefore, employed a quantitative method, western blotting, to study markers of endometrial innervation in the presence and absence of endometriosis. STUDY DESIGN, SIZE, DURATION This study included 45 women undergoing laparoscopic examination for the diagnosis of endometriosis. Endometrial samples were analysed by western blot for the expression of neuronal and neurotrophic markers, PGP9.5, VR1 and NGFp75. PARTICIPANTS/MATERIALS, SETTINGS, METHODS Endometrial pipelle biopsies were obtained from patients with (n = 20, study group) and without (n = 25, control group) endometriosis. Tissue was analysed by immunohistochemistry and western blot analysis for the expression of pan-neuronal marker, PGP9.5, sensory nociceptive marker, TPVR1, and low-affinity neurotrophic growth factor receptor, NGFRp75. MAIN RESULTS AND THE ROLE OF CHANCE PGP9.5, NGFp75 and VR1 were expressed in the endometrium of women, independent of the presence of endometriosis. Furthermore, the expression level of PGP9.5, VR1 and NGFp75 did not alter between the two cohorts of women. LIMITATIONS, REASONS FOR CAUTION Studies of this nature are subject to the heterogeneous nature of patient population and tissue samples despite attempts to standardize these parameters. Hence, further studies using similar methodology will be required to confirm our results. WIDER IMPLICATIONS OF THE FINDINGS Our results highlight that sensory neuronal markers are present in women with and without endometriosis. Future work will assess what the targets of the endometrial nerves are and investigate their function, their impact on endometrial biology and, in particular, whether aberrant neuronal function, rather than the mere presence of neuronal function, could be the root cause of subfertility and/or pain affecting many endometriosis sufferers. Our results do not, however, confirm the previous paradigm of increased innervation in the endometrium of women with endometriosis, nor the use of nerve cell detection from pipelle biopsies to diagnose endometriosis.

FOXP3 immunohistochemistry on formalin-fixed paraffin embedded tissue- poor correlation between different antibodies.

Since its original description, there has been a substantial output of publications related to the FOXP3 gene. The FOXP3 protein, a member of the forkhead/winged-helix family of transcriptional regulators is a nuclear product and is not expressed in the cell cytoplasm or on the cell surface. Expression of this single transcription factor causes a developmental switch in naïve T cells to a suppressor cell phenotype, more commonly referred to as regulatory T cells (Tregs). Tregs have been intensively studied in various autoimmune diseases, infections and different cancers. An increasing choice of commercially available monoclonal antibodies targeting FOXP3 is now available. This report describes the experience of using two commonly used monoclonal FOXP3 antibodies on formalin-fixed paraffin-embedded sections of different organs, including the cervix and vulva. The antibodies targeting different FOXP3 epitopes unexpectedly resulted in significantly different staining patterns. This phenomenon has not been previously reported and is likely to be an important observation.

Danaparoid thromboprophylaxis in pregnant women with heparin‐induced thrombocytopenia

A 38 year old woman presented to the antenatal clinic at 36 weeks of gestation. She had received no antenatal care before this. In her previous nine pregnancies, she had four normal full term vaginal deliveries, two terminations of pregnancy, one miscarriage, one intrauterine death at 36 weeks of gestation and an emergency caesarean delivery at term (her last pregnancy). Nine days after her last delivery, she developed pulmonary embolism. During her treatment with intravenous heparin, she developed thrombocytopenia. Her platelet count decreased gradually from 266 10/L on the second day of treatment to 112 10/L on the fourth day. Platelet factor antibodies were present in her serum and a diagnosis of heparin-induced thrombocytopenia was made. She was anticoagulated with danaparoid (Orgaran-Durbin) and her platelet count recovered within three days. The woman continued anticoagulation with warfarin. Investigations for thrombophilia (protein C, protein S, fibrinogen, factor VIII, lupus anticoagulant, anticardiolipin antibodies, activated protein C ratio and antithrombin) were all negative. Her medical and family histories were otherwise normal. In her index pregnancy the history of heparin-induced thrombocytopenia excluded unfractionated and low molecular weight heparin as thromboprophylactic agents. Because of her past history of thromboembolism, age and multiparity, it was felt that aspirin alone was insufficient as thromboprophylaxis. Therefore, she was treated with aspirin antenatally and danaparoid (750 iu twice daily) was administered subcutaneously during labour and warfarin postnatally. The woman went into spontaneous labour and was delivered spontaneously. During the course of her treatment, she did not develop thrombocytopenia. Both mother and baby were discharged seven days following delivery. She did not breastfeed.