Implementing an Advanced Laparoscopic Procedure by Monitoring with a Visiting Surgeon

Study Objective: To investigate the feasibility of safely implementing a total laparoscopic hysterectomy (LH) in established gynecologists' practices with on-site coaching and monitoring of the learning curve by an experienced visiting surgeon. Design: Multicenter prospective feasibility and implementation study (Canadian Task Force classification II-2). Setting: Eleven general gynecologists in 8 hospitals (1 university hospital and 7 regional hospitals) participated. Patients: Laparoscopic hysterectomy was performed in 83 patients during the learning curve, and in 83 patients after the learning curve. Interventions: During the learning curve, an experienced visiting laparoscopist was available for coaching during each LH. A competence score was marked on an Objective Structured Assessment of Technical Skills (OSATS) form. Complications were recorded intraoperatively and postoperatively for 6 weeks after surgery in all patients. Measurements and Main Results: Nine of 11 gynecologists reached the competence score of at least 28 points during the study, from January 2005 to January 2007. A major complication occurred in 3 of 83 LH procedures (4%) performed during the learning curve, and in 5 of 83 LH procedures (6%) performed after the learning curve (p = .72). Conclusion: The concept of a visiting surgeon for on-site coaching and monitoring of established gynecologists during the learning curve of an advanced laparoscopic procedure using Objectively Structured Assessment of Technical Skills is feasible. According to the observed complication rate during and after the learning curve, on-site coaching is a useful tool when implementing a new laparoscopic technique in established gynecologists' practices. Journal of Minimally Invasive Gynecology (2010) 17, 771-778 (C) 2010 AAGL. All rights reserved

Low-dose triple drug combination targeting the PI3K/AKT/mTOR pathway and the MAPK pathway is an effective approach in ovarian clear cell carcinoma

Advanced stage ovarian clear cell carcinoma (OCCC) is poorly responsive to platinum-based chemotherapy and has an unfavorable prognosis. Previous studies revealed heterogeneous mutations in PI3K/AKT/mTOR and MAPK pathway nodules converging in mTORC1/2 activation. Here, we aimed to identify an effective low-dose combination of PI3K/AKT/mTOR pathway and MAPK pathway inhibitors simultaneously targeting key kinases in OCCC to preclude single-inhibitor initiated pathway rewiring and limit toxicity. Small molecule inhibitors of mTORC1/2, PI3K and MEK1/2 were combined at monotherapy IC20 doses in a panel of genetically diverse OCCC cell lines (n = 7) to determine an optimal low-dose combination. The IC20 dose triple combination reduced kinase activity in PI3K/AKT/mTOR and MAPK pathways, prevented single-inhibitor induced feedback mechanisms and inhibited short and long-term proliferation in all seven cell lines. Finally, this low-dose triple drug combination treatment significantly reduced tumor growth in two genetically characterized OCCC patient-derived xenograft (PDX) models without resulting in weight loss in these mice. The effectiveness and tolerability of this combined therapy in PDX models warrants clinical exploration of this treatment strategy for OCCC and might be applicable to other cancer types with a similar genetic background

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

Comparison of Targeted Mass Spectrometry Techniques With an Immunoassay:A Case Study For HSP90α

PURPOSE: The objective of this study was to better understand factors governing the variability and sensitivity in SRM and PRM, compared to immunoassay. EXPERIMENTAL DESIGN: A 2D-LC-MS/MS-based SRM and PRM assay was developed for quantitative measurements of HSP90α in serum. Forty-three control sera were compared by SRM, PRM and ELISA following the manufacturer's instructions. Serum samples were trypsin-digested and fractionated by SCX chromatography prior to SRM and PRM measurements. Analytical parameters such as linearity, LOD, LOQ, repeatability and reproducibility of the SRM, PRM and ELISA were determined. RESULTS: PRM data obtained by high-resolution mass spectrometry correlated better with ELISA measurements than SRM data measured on a triple quadrupole mass spectrometer. While all three methods (SRM, PRM, ELISA) were able to quantify HSP90α in serum at the ng/mL level, the use of PRM on a high-resolution mass spectrometer reduced variation and showed comparable sensitivity to immunoassay. CONCLUSIONS AND CLINICAL RELEVANCE: Using fractionation, it is possible to measure ng/mL levels of HSP90α in a reproducible, selective and sensitive way using PRM in serum. This opens up the possibility to use PRM in a multiplexed way as an attractive alternative for immunoassays without the use of antibodies or comparable binders. This article is protected by copyright. All rights reserved

Características das espécies que podem influenciar as dinâmicas populacionais de beija-flores na Floresta Atlântica no Sul do Brasil

Orientador : Prof. Dr. James Joseph RoperTese (doutorado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Ecologia e Conservação. Defesa: Curitiba, 18/03/2016Inclui referências : f. 08-14;24-28;56-62;112-120Área de concentração : Ecologia e conservaçãoResumo: A dinâmica populacional em beija-flores na América do Sul é praticamente desconhecida e pouco estudada, devido à dificuldade de captura e, consequentemente, ao baixo número de marcações e recapturas. No entanto, pela diversidade de espécies desta família e sua ampla distribuição, e o papel que desempenha na polinização de recursos efêmeros, entender a sua dinâmica populacional é importante para compreender também a sua ecologia e evolução. Aqui, começamos a preencher esta lacuna através da utilização de estudos de captura-marcação-recaptura para examinar a dinâmica da população e outros aspectos da ecologia de beija-flores. Examinamos a muda de algumas espécies e o dimorfismo sexual e identificação sexual molecular em um grupo de cinco espécies aparentemente monomórficas. Todas as espécies de beija-flores na área de estudo tendem a migrar, mas os detalhes da migração e as comparações foram difíceis de determinar, já que os indivíduos marcados não foram recapturados longe da nossa área. A espécie mais abundante e consistentemente comum foi o beija-flor-de-papo-branco (Leucochloris albicollis), com tamanho populacional variando aproximadamente entre 150-450 indivíduos (variação mensal), e com o pico de abundância em março, após a aparente temporada de reprodução entre novembro e dezembro. A sobrevivência para esta espécie foi estimada em 33% ao ano, o que é baixa para uma ave tropical. O padrão da muda das penas primárias de voo em nossa assembleia é semelhante ao já registrado em outras assembleias de beija-flores, no sentido proximal para o distal. Entretanto, em nossas espécies houve menos variação na sequência de troca das penas mais distais em comparação aos outros estudos. A muda das penas secundárias foi mais variável na sequência de troca, enquanto as retrices foram mais consistentes. O período de muda iniciou-se a partir de dezembro e se estendeu até meados de abril, mas determinar corretamente a duração do período foi difícil, uma vez que alguns indivíduos migraram enquanto realizavam a muda, delimitando a recaptura para determinar o período concreto. Três das cinco espécies aparentemente monomórficas - uma vez que o sexo foi identificado através de nossa nova técnica molecular para beija-flores - são sexualmente dicromáticas (Colibri serrirostris, Eupetomena macroura, L. albicollis) na reflexão de luz UV em suas penas. O tamanho e forma, no entanto, foram semelhantes em ambos os sexos para todas as espécies. Estes resultados demonstram que algumas espécies de beija-flores podem ser muito abundantes, mas que esta abundância pode variar amplamente ao longo do ano. Também, mostramos a existência de migração durante a muda, porém sem detalhamentos sobre a duração total deste período, permanecendo esta questão ainda desconhecida. As taxas de sobrevivência foram baixas para L. albicollis comparadas à outras espécies de aves tropicais, sugerindo relativamente alto sucesso reprodutivo. Por fim, mostramos dicromatismo sexual à luz UV (imperceptível aos seres humanos) em três das cinco espécies, sugerindo que pode haver também nas demais, mas que precisa ser descoberto em uma análise mais aprofundada das penas de outras regiões do corpo. Assim, com este estudo nós iniciamos um melhor entendimento das dinâmicas populacionais em beija-flores. Palavras-chave: coloração, dimorfismo sexual, dinâmicas populacionais, morfometria, muda das penas de voo, sexagem, TrochilidaeAbstract: Population dynamics in hummingbirds in South America is virtually unknown and unstudied, due to difficulty in their capture in numbers for mark and recapture studies. Yet, due to species diversity, distributions, the role they play as pollinators of ephemeral resources and their colorful displays, study of their population dynamics is important for understanding their ecology and evolution. Here, we begin to fill this lacuna by using capture-mark-recapture to examine population dynamics, and other aspects of hummingbird ecology. We examined molt in a variety of species and sexual dimorphism and molecular identification of the sexes in a group of five apparently monomorphic species. All hummingbird species in the study area tend to migrate, but details of migration and comparisons are difficult to determine because marked birds were never recaptured away from the study area. The most abundant and consistently common species, the Whitethroated Hummingbird (Leucochloris albicollis) had a population size that varied from ~150-450 individuals (monthly variation) in the study area, with the peak abundance in March, following after the apparent breeding season in November-December. Survival was estimated at 33% per year, which is low for a tropical bird. Molt is similar to that of other hummingbirds, from proximal to distal primary feathers, but in our study species varied less in the sequence of the most distal feathers as compared to other studies. Secondaries were quite variable in their sequence of molt, while retrices were more consistent. Molt took place beginning in December and continued to April, but defining the end of molt was difficult because species migrate while undergoing molt, and so recaptures to delimit the end of molt was not possible. Three of the five apparently monomorphic species, once sex was identified through our new molecular technique for hummingbirds, were sexually dichromatic (Colibri serrirostris, Eupetomena macroura, L. albicollis) in UV light reflectance in their feathers. Size and shape, however, were similar in both sexes. With these results, we find that hummingbird species can be very abundant, but abundance varies widely over the year, they migrate while molting but the details of their end points of migration remain unknown, and survival rates are low, suggesting relatively high reproductive success. We show sexual dichromatism in UV light (which humans do not perceive) in three of five species, and suggest that the others also are dichromatic, which will be discovered on further examination of the appropriate feathers. Thus, with this study we have begun to better understand hummingbird population dynamics. Keywords: coloration, molting of flight feathers, morphometry, population dynamics, sexing, sexual dimorphism, Trochilida

Folate Receptor-Beta Has Limited Value for Fluorescent Imaging in Ovarian, Breast and Colorectal Cancer

Aims Tumor-specific targeted imaging is rapidly evolving in cancer diagnosis. The folate receptor alpha (FR-alpha) has already been identified as a suitable target for cancer therapy and imaging. FR-alpha is present on similar to 40% of human cancers. FR-beta is known to be expressed on several hematologic malignancies and on activated macrophages, but little is known about FR-beta expression in solid tumors. Additional or simultaneous expression of FR-beta could help extend the indications for folate-based drugs and imaging agents. In this study, the expression pattern of FR-beta is evaluated in ovarian, breast and colorectal cancer. Methods FR-beta expression was analyzed by semi-quantitative scoring of immunohistochemical staining on tissue microarrays (TMAs) of 339 ovarian cancer patients, 418 breast cancer patients, on 20 slides of colorectal cancer samples and on 25 samples of diverticulitis. Results FR-beta expression was seen in 21% of ovarian cancer samples, 9% of breast cancer samples, and 55% of colorectal cancer samples. Expression was weak or moderate. Of the diverticulitis samples, 80% were positive for FR-beta expression in macrophages. FR-beta status neither correlated to known disease-related variables, nor showed association with overall survival and progression free survival in ovarian and breast cancer. In breast cancer, negative axillary status was significantly correlated to FR-beta expression (p=0.022). Conclusions FR-beta expression was low or absent in the majority of ovarian, breast and colorectal tumor samples. From the present study we conclude that the low FR-beta expression in ovarian and breast tumor tissue indicates limited practical use of this receptor in diagnostic imaging and therapeutic purposes. Due to weak expression, FR-beta is not regarded as a suitable target in colorectal cancer

Biobanking of patient and patient-derived xenograft ovarian tumour tissue:efficient preservation with low and high fetal calf serum based methods

Using patient-derived xenografts (PDXs) for preclinical cancer research demands proper storage of tumour material to facilitate logistics and to reduce the number of animals needed. We successfully established 45 subcutaneous ovarian cancer PDXs, reflecting all histological subtypes, with an overall take rate of 68%. Corresponding cells from mouse replaced human tumour stromal and endothelial cells in second generation PDXs as demonstrated with mouse-specific vimentin and CD31 immunohistochemical staining. For biobanking purposes two cryopreservation methods, a fetal calf serum (FCS)-based (95% v/v) "FCS/DMSO" protocol and a low serum-based (10% v/v) "vitrification" protocol were tested. After primary cryopreservation, tumour take rates were 38% and 67% using either the vitrification or FCS/DMSO-based cryopreservation protocol, respectively. Cryopreserved tumour tissue of established PDXs achieved take rates of 67% and 94%, respectively compared to 91% using fresh PDX tumour tissue. Genotyping analysis showed that no changes in copy number alterations were introduced by any of the biobanking methods. Our results indicate that both protocols can be used for biobanking of ovarian tumour and PDX tissues. However, FCS/DMSO-based cryopreservation is more successful. Moreover, primary engraftment of fresh patient-derived tumours in mice followed by freezing tissue of successfully established PDXs is the preferred way of efficient ovarian cancer PDX biobanking

Kinome capture sequencing of high-grade serous ovarian carcinoma reveals novel mutations in theJAK3gene

High-grade serous ovarian carcinoma (HGSOC) remains the deadliest form of epithelial ovarian cancer and despite major efforts little improvement in overall survival has been achieved. Identification of recurring "driver" genetic lesions has the potential to enable design of novel therapies for cancer. Here, we report on a study to find such new therapeutic targets for HGSOC using exome-capture sequencing approach targeting all kinase genes in 127 patient samples. Consistent with previous reports, the most frequently mutated gene wasTP53(97% mutation frequency) followed byBRCA1(10% mutation frequency). The average mutation frequency of the kinase genes mutated from our panel was 1.5%. Intriguingly, afterBRCA1,JAK3was the most frequently mutated gene (4% mutation frequency). We tested the transforming properties of JAK3 mutants using the Ba/F3 cell-basedin vitrofunctional assay and identified a novel gain-of-function mutation in the kinase domain ofJAK3(p.T1022I). Importantly, p.T1022IJAK3mutants displayed higher sensitivity to the JAK3-selective inhibitor Tofacitinib compared to controls. For independent validation, we re-sequenced the entireJAK3coding sequence using tagged amplicon sequencing (TAm-Seq) in 463 HGSOCs resulting in an overall somatic mutation frequency of 1%. TAm-Seq screening ofCDK12in the same population revealed a 7% mutation frequency. Our data confirms that the frequency of mutations in kinase genes in HGSOC is low and provides accurate estimates for the frequency ofJAK3andCDK12mutations in a large well characterized cohort. Although p.T1022IJAK3mutations are rare, our functional validation shows that if detected they should be considered as potentially actionable for therapy. The observation ofCDK12mutations in 7% of HGSOC cases provides a strong rationale for routine somatic testing, although more functional and clinical characterization is required to understand which nonsynonymous mutations alterations are associated with homologous recombination deficiency.ISSN:1932-620

The role of ATM and 53BP1 as predictive markers in cervical cancer

Treatment of advanced-stage cervical cancers with (chemo)radiation causes cytotoxicity through induction of high levels of DNA damage. Tumour cells respond to DNA damage by activation of the DNA damage response (DDR), which induces DNA repair and may counteract chemoradiation efficacy. Here, we investigated DDR components as potential therapeutic targets and verified the predictive and prognostic value of DDR activation in patients with cervical cancer treated with (chemo)radiation. In a panel of cervical cancer cell lines, inactivation of ataxia telangiectasia mutated (ATM) or its substrate p53-binding protein-1 (53BP1) clearly gave rise to cell cycle defects in response to irradiation. Concordantly, clonogenic survival analysis revealed that ATM inhibition, but not 53BP1 depletion, strongly radiosensitised cervical cancer cells. In contrast, ATM inhibition did not radiosensitise non-transformed epithelial cells or non-transformed BJ fibroblasts. Interestingly, high levels of active ATM prior to irradiation were related with increased radioresistance. To test whether active ATM in tumours prior to treatment also resulted in resistance to therapy, immunohistochemistry was performed on tumour material of patients with advanced-stage cervical cancer (n = 375) treated with (chemo)radiation. High levels of phosphorylated (p-)ATM [p = 0.006, hazard ratio (HR) = 1.817] were related to poor locoregional disease-free survival. Furthermore, high levels of p-ATM predicted shorter disease-specific survival (p = 0.038, HR = 1.418). The presence of phosphorylated 53BP1 was associated with p-ATM (p = 0.001, odds ratio = 2.206) but was not related to any clinicopathological features or survival. In conclusion, both our in vitro and patient-related findings indicate a protective role for ATM in response to (chemo)radiation in cervical cancer and point at ATM inhibition as a possible means to improve the efficacy of (chemo)radiation