Factors associated with human papillomavirus persistence after loop electrosurgical excision procedure in patients with cervical squamous intraepithelial lesion.

AIM: To seek the high-risk factors of human papillomavirus (HPV) persistence and residual lesion or recurrence after loop electrosurgical excision procedure (LEEP) focus on the predictive value of intraoperative human papilloma virus (IOP-HPV) testing. METHODS: Intraoperative endocervical sample was obtained with a cytobrush from the remained cervix of 292 patients immediately after LEEP. HPV Genotyping was performed using a polymerase chain reaction technique. All patients followed by HPV genotyping and cytology every 3-6 months. The IOP-HPV testing results and possible risk factors such as age, cytology grade, menopause status, margin involvement, preoperative HPV status, and cervical lesion grade were assessed in predicting persistence of HPV and residual lesion or recurrence after surgery. RESULTS: There were 61 (20.9%) patients presented persistent HPV infection. Multivariate analyses showed that IOP-HPV positive, post-menopause and preoperative HPV multiplex infection was strongly associated with HPV persistence after LEEP, IOP-HPV positive and post-menopause was also associated with residua or recurrent disease after LEEP. CONCLUSIONS: IOP-HPV positive, post-menopause, and preoperative HPV multiplex infection are independent predictors of HPV persistence in patients with cervical squamous intraepithelial lesion treated by LEEP. IOP-HPV test is a new approach that may potentially allow for early identification of patients at high risk of HPV persistence and residua or recurrent disease after LEEP, thereby possibly facilitate an attenuated follow-up schedule for negative patients those at low risk of persistent HPV infection.

Design of a palette of SNAP-tag mimics of fluorescent proteins and their use as cell reporters.

Naturally occurring fluorescent proteins (FPs) are the most widely used tools for tracking cellular proteins and sensing cellular events. Here, we chemically evolved the self-labeling SNAP-tag into a palette of SNAP-tag mimics of fluorescent proteins (SmFPs) that possess bright, rapidly inducible fluorescence ranging from cyan to infrared. SmFPs are integral chemical-genetic entities based on the same fluorogenic principle as FPs, i.e., induction of fluorescence of non-emitting molecular rotors by conformational locking. We demonstrate the usefulness of these SmFPs in real-time tracking of protein expression, degradation, binding interactions, trafficking, and assembly, and show that these optimally designed SmFPs outperform FPs like GFP in many important ways. We further show that the fluorescence of circularly permuted SmFPs is sensitive to the conformational changes of their fusion partners, and that these fusion partners can be used for the development of single SmFP-based genetically encoded calcium sensors for live cell imaging.

Analysis of systemic inflammatory and coagulation biomarkers in advanced cervical cancer: Prognostic and predictive significance.

OBJECTIVE: Peripheral systemic inflammatory, nutritional, and coagulation biomarkers have prognostic and predictive value in various malignancies. We evaluated the prognostic and predictive roles of systemic inflammatory, nutritional, and coagulation biomarkers in the circulating blood of patients with advanced cervical cancer. METHODS: A retrospective study of 795 patients with cervical cancer who received concurrent chemoradiation therapy was performed. Overall survival was evaluated by the Kaplan-Meier estimator. Univariate and multivariate Cox regression models were used to determine prognostic factors associated with overall survival. RESULTS: The median follow-up time was 76 months. In the univariate analysis, overall survival showed positive prognostic value in patients with a platelet-to-lymphocyte ratio (PLR) <164.29 (P = 0.010), and a plasma fibrinogen (FIB) level <4 g/L(P = 0.012). In the multivariate analysis, the PLR (P = 0.036), and FIB level (P = 0.047) maintained their significance for overall survival. Therefore, the PLR and FIB levels are independent prognostic factors in patients with advanced cervical cancer. CONCLUSIONS: Systemic inflammatory and coagulation biomarkers could help to understand survival differences in the clinical treatment of advanced cervical cancer. The PLR and FIB levels are independent prognostic factors of poor survival in patients with advanced cervical cancer.

Tolerance and Heavy Metal Accumulation Characteristics of Sasa argenteostriata (Regel) E.G. Camus under Zinc Single Stress and Combined Lead-Zinc Stress.

Sasa argenteostriata (Regel) E.G. Camus is a gramineous plant with the potential for phytoremediation. In this study, we aimed to determine its tolerance to zinc stress and combined lead-zinc stress and the effect of zinc on its absorption and accumulation characteristics of lead. The results showed that S. argenteostriata had good tolerance to zinc stress, and S. argenteostriata was not significantly damaged when the zinc stress concentration was 600 mg/L. Under both zinc stress and combined lead-zinc stress, the root was the main organ that accumulated heavy metals in S. argenteostriata. The presence of zinc promoted the absorption of lead by the root of S. argenteostriata, and the lead content in the root under PZ1, PZ2, PZ3 and PZ4 treatments was 2.15, 4.31, 4.47 and 6.01 times that of PZ0 on the 20 days. In the combined lead-zinc stress treatments, the toxicity of heavy metals to S. argenteostriata was mainly caused by lead. Under high concentrations of combined lead-zinc stress (PZ4), the proportion of zinc in the leaf of S. argenteostriata on the 20 days increased, which was used as a tolerance strategy to alleviate the toxicity of lead.

Comparison of different treatment strategies in the management of endogenic caesarean scar pregnancy: a multicentre retrospective study.

BACKGROUND: The aim of this study was to evaluate the effectiveness and safety of different treatment strategies for endogenic caesarean scar pregnancy (CSP) patients. METHODS: According to Vial's standard, we defined endogenic-type CSP as (1) the gestational sac growing towards the uterine cavity and (2) a greater than 0.3 cm thickness of myometrial tissue at the caesarean scar. A total of 447 endogenic CSP patients out of 527 patients from 4 medical centres in China were enrolled in this study. A total of 120 patients were treated with methotrexate (MTX) followed by surgery, 106 received ultrasound-guided curettage directly and 221 received curettage combined with hysteroscopy. The clinical information and clinical outcomes of these patients were reviewed. Successful treatment was defined as (1) no additional treatment needed, (2) no retained mass of conception and (3) serum ß subunit of human chorionic gonadotropin (ß-hCG) level returning to a normal level within 4 weeks. The success rate was analysed based on these factors. RESULT: Among 447 patients, no significant difference was observed in baseline characteristics between groups except for foetal heartbeat. The success rate was significantly different (p<0.001) among the three groups. The highest success rate of 95.9% was noted in the hysteroscopy group, and the lowest success rate of 84.0% was noted in the curettage group. In addition, the MTX group reported the longest hospital stay and highest expenses, but the curettage group showed the shortest and lowest expenses, respectively. Nevertheless, no difference in blood loss was observed between the groups. CONCLUSION: The combination of curettage and hysteroscopy represents the most effective strategy. Pretreatment with MTX did not result in better clinical outcomes. Ultrasound-guided curettage directly should not be considered a first-line treatment choice for endogenic CSP patients.

Biomass allocation strategies and Pb-enrichment characteristics of six dwarf bamboos under soil Pb stress.

Dwarf bamboos are clonal plants with potential applications in the remediation of heavy metal-polluted soils, although their pollution adaptation strategies are unknown. This study examined the biomass allocation strategies and lead (Pb) enrichment characteristics of various dwarf bamboo tissues by the end of the growing season and explored their potential for phytoremediation of Pb stress in the soils. Six dwarf bamboo genotypes were treated with three levels (0, 300, and 1500 mg kg-1) of soil Pb stress. The majority of the bamboos adopted two biomass allocation strategies to adapt to Pb stress, namely, "reducing biomass allocation into new bamboo growth" and "increasing/stabilizing biomass allocation into rhizomes". Pb accumulation was highest in the roots, rhizomes, and old stems and showed the following trend: rhizomes/old stems> new roots/old roots> old leaves> new leaves> new stems among various tissues. Moreover, the six bamboos used three different Pb-enrichment strategies, as follows: (i) "rhizome domination and old stem synergy" (Sasaella glabra (Nakai) f. albo-striata Muroi, Sasa auricoma (Mitford) E.G. Camus, Sasa fortunei (Van Houtte) Fiori, and Shibataea lanceifolia C.H. Hu); (ii) "old stem domination and rhizome synergy" (Indocalamus decorus Q.H. Dai); and (iii) "old stem domination and new root synergy" (Sasa argenteostriata (Regel) E.G. Camus). In Pb-contaminated soils, genotypes with TFs greater than 1 were Sasa fortunei (Van Houtte) Fiori, Sasa argenteostriata (Regel) E.G. Camus, and Indocalamus decorus Q.H. Dai; in addition, only S. argenteostriata had BCF values greater than 1. Furthermore, this study provides the first evidence that S. argenteostriata can extract 0.22 and 0.58 mgplant-1 of Pb ions in soil polluted with 300 and 1500 mg kg-1 Pb, respectively. S. argenteostriata showed the greatest potential for phytoremediation among the bamboo genotypes in both Pb-contaminated urban and mining sites.

Topical administration of rapamycin promotes retinal ganglion cell survival and reduces intraocular pressure in a rat glaucoma model.

Glaucoma is a progressive optic neuropathy that has become the most common cause of irreversible blindness worldwide. Studies have shown that the protein mammalian target of rapamycin (mTOR) is a serine/threonine kinase that plays a central role in regulating numerous functions, such as growth, proliferation, cytoskeletal organization, metabolism, and autophagy. Clinical trials have shown that Rho-associated protein kinase (ROCK) inhibitors reduced intraocular pressure (IOP) in patients with glaucoma and ocular hypertension (OHT). In this study, we explored whether rapamycin (RAPA) eye drops can reduce IOP and protect retinal ganglion cells (RGCs). Our results indicated that in rats treated with RAPA, the drug was detected in the aqueous humor (AH), and the IOP was reduced. This may be related to the inhibition of RhoA protein activation by RAPA and regulation of the actin cytoskeleton in trabecular meshwork (TM) cells. In addition, the retinal thickness and the survival rate of RGCs were significantly reduced in the OHT group compared with the control group. These changes in the OHT group were significantly improved after treatment with RAPA. This may be because RAPA inhibited the activation of glial cells and the release of proinflammatory factors, thereby attenuating further damage to the retina and RGCs. Taken together, the results of this study demonstrated that RAPA not only reduced IOP but also protected RGCs, suggesting that RAPA is likely to be an effective strategy for the treatment of glaucoma.

Development and Validation of a Personalized Survival Prediction Model for Uterine Adenosarcoma: A Population-Based Deep Learning Study.

BACKGROUND: The aim was to develop a personalized survival prediction deep learning model for adenosarcoma patients using the surveillance, epidemiology and end results (SEER) database. METHODS: A total of 797 uterine adenosarcoma patients were enrolled in this study. Duplicated and useless variables were excluded, and 15 variables were selected for further analyses, including age, grade, positive lymph nodes or not, marital status, race, tumor extension, stage, and surgery or not. We created our deep survival learning (DSL) model to manipulate the data, which was randomly split into a training set (n = 519, 65%), validation set (n = 143, 18%) and testing set (n = 143, 18%). The Cox proportional hazard (CPH) model was also included comparatively. Finally, personalized survival curves were plotted for randomly selected patients. RESULTS: The c-index for the CPH model was 0.726, and the Brier score was 0.17. For our deep survival learning model, we achieved a c-index of 0.774 and a Brier score of 0.14 in the external testing set. In addition, the limitations of the traditional staging system were revealed, and a personalized survival prediction system based on our risk scoring grouping was developed. CONCLUSIONS: Our study developed a deep neural network model for adenosarcoma. The performance of this model was superior to that of the traditional Cox proportional hazard model. In addition, a personalized survival prediction system was developed based on our deep survival learning model, which provided more accurate prognostic information for adenosarcoma patients.

Visualizing RNA dynamics in live cells with bright and stable fluorescent RNAs.

Fluorescent RNAs (FRs), aptamers that bind and activate fluorescent dyes, have been used to image abundant cellular RNA species. However, limitations such as low brightness and limited availability of dye/aptamer combinations with different spectral characteristics have limited use of these tools in live mammalian cells and in vivo. Here, we develop Peppers, a series of monomeric, bright and stable FRs with a broad range of emission maxima spanning from cyan to red. Peppers allow simple and robust imaging of diverse RNA species in live cells with minimal perturbation of the target RNA's transcription, localization and translation. Quantification of the levels of proteins and their messenger RNAs in single cells suggests that translation is governed by normal enzyme kinetics but with marked heterogeneity. We further show that Peppers can be used for imaging genomic loci with CRISPR display, for real-time tracking of protein-RNA tethering, and for super-resolution imaging. We believe these FRs will be useful tools for live imaging of cellular RNAs.

Resveratrol Delays Retinal Ganglion Cell Loss and Attenuates Gliosis-Related Inflammation From Ischemia-Reperfusion Injury.

Purpose: Resveratrol has been shown to enhance the survival of retinal ganglion cells (RGCs) following ischemia-reperfusion (I/R) injury for glaucoma. However, the precise mechanisms for resveratrol's protective effects are still unclear. The aim of this study is to determine whether resveratrol can inhibit RGC apoptosis, retinal gliosis, and inflammation, all of which are critical events in retinal degeneration following I/R injury. Methods: Right retinal ischemia was induced in adult male Sprague Dawley rats by increasing intraocular pressure to 110 mm Hg for 60 minutes, and the left eyes maintained at normal pressure serve as the control. Intraperitoneal injection of resveratrol or control buffer was performed continuously for 3 days from pre- to post-I/R injury and the protective effects were evaluated and compared. RGCs were retrogradely labeled with Fluoro-Gold by injection into superior colliculi. Apoptosis was detected by TUNEL staining. Western blotting and immunostaining for Bax, Bcl-2, and Caspase-3 were used to explore the Bax-associated apoptotic pathway. Gliosis was assessed by western blotting and immunostaining of retinal cross sections with anti-glial fibrillary acidic protein (GFAP) antibodies. Results: In this study, resveratrol treatment significantly reduced retinal damage and RGC loss as demonstrated by the relatively intact tissue structure in hematoxylin and eosin staining at day 7 and increased Fluoro-Gold labeling of RGCs at day 14, respectively. We found that resveratrol exhibited an anti-apoptotic effect as assessed by reduced TUNEL staining, inhibition of the early upregulated expression of the apoptosis-related protein Bax, and decreased subsequently cleaved caspase-3. However, it did not affect Bcl-2 levels. Moreover, in our I/R injury model, the combined response of reactive gliosis and related inflammation, which were demonstrated by an early induction of pro-inflammatory mediators and subsequently increased GFAP level, were significantly attenuated after resveratrol treatment. Conclusions: These results demonstrate that resveratrol can prevent RGC death by blocking the Bax-caspase-3-dependent apoptotic pathway and suppressed gliosis-related inflammation in the retina after I/R injury. Together these results support the use of resveratrol as a possible therapeutic strategy for glaucoma.

Rapamycin mediates mTOR signaling in reactive astrocytes and reduces retinal ganglion cell loss.

Damage and loss of retinal ganglion cells (RGCs) can cause visual impairment. The underlying molecular mechanisms that mediate RGC death in ischemic retinal diseases are still unclear. In this study, we sought to understand the neuroprotective effect of rapamycin, the selective inhibitor of mTORC1, on RGC survival and the cellular mechanics that mediate this effect. Recent studies have reported that the epidermal growth factor (EGF) receptor shows an increase in expression in astrocytes after injury, and this receptor can promote their transformation into reactive astrocytes. Our results, along with previous works from others, show the colocalization of phosphor-EGF receptors with the astrocyte marker glial fibrillary acidic proteins in reactive astrocytes in the injured retina. In our in vitro studies, using primary astrocyte cultures of the optic nerve head of rats, showed that rapamycin significantly blocked EGF-induced mTOR signaling mainly through the PI3K/Akt pathway in primary astrocytes, but not through the MAPK/Erk pathway. Additionally, rapamycin dramatically inhibited the activation of mTOR signaling in our ratinal ischemia-reperfusion (I/R) injury model in vivo. Astrocyte activation was assessed by immunostaining retinal flat mounts or cross sections with antibody against GFAP, and we also used western blots to detect the expression of GFAP. Taken together, these results revealed that rapamycin decreases the activation of astrocytes after retinal ischemia-reperfusion injury. Furthermore, rapamycin can improve retinal RGC survival in rats during I/R, as detected by FluoroGold labeling. Our data reveals the neuroprotective effects of rapamycin in an experimental retina injury model, possibly through decreasing glial-dependent intracellular signaling mechanisms for suppressing apoptosis of RGCs. Our study also presents an approach to targeting reactive astrocytes for the treatment of optic neurodegenerations.

Physiological pH-dependent gelation for 3D printing based on the phase separation of gelatin and oxidized dextran.

An extrudable hydrogel with a tunable gelation time under physiological pH ranges based on the phase separation of gelatin and oxidized dextran was demonstrated. We envision that the easy handing properties of this hydrogel combined with thermosensitive physical gelation and postponed chemical reinforcing will provide a platform for 3D bioprinting applications.

RhoC interacts with integrin α5ß1 and enhances its trafficking in migrating pancreatic carcinoma cells.

Human pancreatic ductal adenocarcinoma (PDAC) is characterized by early systemic dissemination. Although RhoC has been implicated in cancer cell migration, the relevant underlying molecular mechanisms remain unknown. RhoC has been implicated in the enhancement of cancer cell migration and invasion, with actions which are distinct from RhoA (84% homology), and are possibly attributed to the divergent C-terminus domain. Here, we confirm that RhoC significantly enhances the migratory and invasive properties of pancreatic carcinoma cells. In addition, we show that RhoC over-expression decreases cancer cell adhesion and, in turn, accelerates cellular body movement and focal adhesion turnover, especially, on fibronectin-coated surfaces. Whilst RhoC over-expression did not alter integrin expression patterns, we show that it enhanced integrin α5ß1 internalization and re-cycling (trafficking), an effect that was dependent specifically on the C-terminus (180-193 amino acids) of RhoC protein. We also report that RhoC and integrin α5ß1 co-localize within the peri-nuclear region of pancreatic tumor cells, and by masking the CAAX motif at the C-terminal of RhoC protein, we were able to abolish this interaction in vitro and in vivo. Co-localization of integrin α5ß1 and RhoC was demonstrable in invading cancer cells in 3D-organotypic cultures, and further mimicked in vivo analyses of, spontaneous human, (two distinct sources: operated patients and rapid autopsy programme) and transgenic murine (LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre), pancreatic cancers. In both cases, co-localization of integrin α5ß1 and RhoC correlated with poor differentiation status and metastatic potential. We propose that RhoC facilitates tumor cell invasion and promotes subsequent metastasis, in part, by enhancing integrin α5ß1 trafficking. Thus, RhoC may serve as a biomarker and a therapeutic target.

The association of CCND1 overexpression and cisplatin resistance in testicular germ cell tumors and other cancers.

Development of chemoresistance limits the clinical efficiency of platinum-based therapy. Although many resistance mechanisms have been demonstrated, genetic/molecular alterations responsible for drug resistance in the majority of clinical cases have not been identified. We analyzed three pairs of testicular germ cell tumor cell lines using Affymetrix expression microarrays and revealed a limited number of differentially expressed genes across the cell lines when comparing the parental and resistant cells. Among them, CCND1 was the most significantly differentially expressed gene. Analysis of testicular germ cell tumor clinical samples by quantitative reverse transcription PCR analysis revealed that overall expression of CCND1 was significantly higher in resistant cases compared with sensitive samples (P < 0.0001). We also found that CCND1 was dramatically overexpressed both in induced and intrinsically resistant samples of ovarian and prostate cancer. Finally combined CCND1 knockdown using small-interfering RNA and cisplatin treatment inhibited cell growth in vitro significantly more effectively than any of these single treatments. Therefore, deregulation of CCND1 may be a major cause of cisplatin resistance in testicular germ cell tumors and may also be implicated in ovarian and prostate cancers. CCND1 could be potentially used as a marker for treatment stratification and as a molecular target to improve the treatment of platinum-resistant tumors.

Tumor necrosis factor alpha as a new target for renal cell carcinoma: two sequential phase II trials of infliximab at standard and high dose.

PURPOSE: Tumor necrosis factor alpha (TNF-alpha) may play a role in renal cell carcinoma (RCC). We performed two sequential phase II studies of infliximab, an anti-TNF-alpha monoclonal antibody, in patients with immunotherapy-resistant or refractory RCC. PATIENTS AND METHODS: Patients progressing after cytokine therapy were treated with intravenous infliximab as follows: study 1 (19 patients), 5 mg/kg at weeks 0, 2, and 6, and then every 8 weeks; study 2 (18 patients), 10 mg/kg at weeks 0, 2, and 6, and then every 4 weeks. Treatment continued until disease progression (PD). Response was assessed according to Response Evaluation Criteria in Solid Tumors. Plasma levels of TNF-alpha, CCL2, and interleukin-6 (IL-6) were measured before and during treatment. RESULTS: TNF-alpha and its receptors were detected in malignant cells in RCC biopsies. In study 1, three patients (16%) achieved partial response (PR) and three patients (16%) achieved stable disease (SD). Median duration of response (PR + SD) was 7.7 months (range, 5.0 to 40.5+ months). In study 2, 11 patients (61%) achieved SD. Median duration of response was 6.2 months (range, 3.5 to 24+ months). One patient developed grade 3 hypersensitivity and another died as a result of pulmonary infection/sepsis. Enzyme-linked immunosorbent assay analysis of plasma revealed that higher levels of TNF-alpha at baseline and higher levels of CCL2 during treatment were associated with PD. There were also correlations between higher levels of TNF-alpha, IL-6, and CCL2 and poor survival (< 12 months). CONCLUSION: This is the first direct clinical evidence suggesting that TNF-alpha may be a therapeutic target in RCC. Plasma levels of TNF-alpha, IL-6, and CCL2 may have predictive and prognostic significance.

Macrophages induce invasiveness of epithelial cancer cells via NF-kappa B and JNK.

Tumor-associated macrophages may influence tumor progression, angiogenesis and invasion. To investigate mechanisms by which macrophages interact with tumor cells, we developed an in vitro coculture model. Previously we reported that coculture enhanced invasiveness of the tumor cells in a TNF-alpha- and matrix metalloprotease-dependent manner. In this report, we studied intracellular signaling pathways and induction of inflammatory genes in malignant cells under the influence of macrophage coculture. We report that coculture of macrophages with ovarian or breast cancer cell lines led to TNF-alpha-dependent activation of JNK and NF-kappaB pathways in tumor cells, but not in benign immortalized epithelial cells. Tumor cells with increased JNK and NF-kappaB activity exhibited enhanced invasiveness. Inhibition of the NF-kappaB pathway by TNF-alpha neutralizing Abs, an NF-kappaB inhibitor, RNAi to RelA, or overexpression of IkappaB inhibited tumor cell invasiveness. Blockade of JNK also significantly reduced invasiveness, but blockade of p38 MAPK or p42 MAPK had no effect. Cocultured tumor cells were screened for the expression of 22 genes associated with inflammation and invasion that also contained an AP-1 and NF-kappaB binding site. EMMPRIN and MIF were up-regulated in cocultured tumor cells in a JNK- and NF-kappaB-dependent manner. Knocking down either MIF or EMMPRIN by RNAi in the tumor cells significantly reduced tumor cell invasiveness and matrix metalloprotease activity in the coculture supernatant. We conclude that TNF-alpha, via NF-kappaB, and JNK induces MIF and EMMPRIN in macrophage to tumor cell cocultures and this leads to increased invasive capacity of the tumor cells.

A modified medium that significantly improves the growth of human normal ovarian surface epithelial (OSE) cells in vitro.

Approximately 90% of malignant ovarian tumours are epithelial and thought to arise from a single cell layer, the ovarian surface epithelium. In culture, human normal ovarian surface epithelial (OSE) cells have a very limited lifespan before they senesce, rarely progressing beyond 10 population doublings. This has restricted the use of normal OSE cells for studying the biology of ovarian surface epithelium and identifying molecular events that contribute to malignant transformation. We have investigated the conditions for culturing human, normal OSE cells in vitro using modified media. Culturing normal OSE cells in a modified medium (NOSE-CM) supplemented with epidermal growth factor, hydrocortisone, insulin and bovine pituitary extract led to significant improvements in the seeding and cloning efficiencies, overall cell growth and lifespan compared to culturing in a basic, nonsupplemented medium (BM) and previously used media (F-12 K medium and William's medium E). Cells cultured in NOSE-CM underwent, on an average, 19.0 population doublings (95% CI 16.3-21.7); cells cultured in BM underwent 0.43-3.52 population doublings over a similar time period. Growth curves established for different lines indicated that OSE cells continued to grow beyond passage 11 and up to passage 18 in NOSE-CM, but never beyond passage 7 when cultured in BM. It is likely that establishing optimal conditions for the growth of OSE cells in vitro will enable studies of the biological and genetic mechanisms of transformation in epithelial ovarian cancers.