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SummaryMesendodermal specification is one of the earliest events in embryogenesis, where cells first acquire distinct identities. Cell differentiation is a highly regulated process that involves the function of numerous transcription factors (TFs) and signaling molecules, which can be described with gene regulatory networks (GRNs). Cell differentiation GRNs are difficult to build because existing mechanistic methods are low-throughput, and high-throughput methods tend to be non-mechanistic. Additionally, integrating highly dimensional data comprised of more than two data types is challenging. Here, we use linked self-organizing maps to combine ChIP-seq/ATAC-seq with temporal, spatial and perturbation RNA-seq data fromXenopus tropicalismesendoderm development to build a high resolution genome scale mechanistic GRN. We recovered both known and previously unsuspected TF-DNA/TF-TF interactions and validated through reporter assays. Our analysis provides new insights into transcriptional regulation of early cell fate decisions and provides a general approach to building GRNs using highly-dimensional multi-omic data sets.HighlightsBuilt a generally applicable pipeline to creating GRNs using highly-dimensional multi-omic data setsPredicted new TF-DNA/TF-TF interactions during mesendoderm developmentGenerate the first genome scale GRN for vertebrate mesendoderm and expanded the core mesendodermal developmental network with high fidelityDeveloped a resource to visualize hundreds of RNA-seq and ChIP-seq data using 2D SOM metaclusters.

Mitochondrial biogenesis and function are controlled by anterograde regulatory pathways involving more than 1000 nuclear-encoded proteins. Transcriptional networks controlling the nuclear-encoded mitochondrial genes remain to be fully elucidated. Here, we show that histone demethylase LSD1 KO from adult mouse liver (LSD1-LKO) reduces the expression of one-third of all nuclear-encoded mitochondrial genes and decreases mitochondrial biogenesis and function. LSD1-modulated histone methylation epigenetically regulates nuclear-encoded mitochondrial genes. Furthermore, LSD1 regulates gene expression and protein methylation of nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1), which controls the final step of NAD+ synthesis and limits NAD+ availability in the nucleus. Lsd1 KO reduces NAD+-dependent SIRT1 and SIRT7 deacetylase activity, leading to hyperacetylation and hypofunctioning of GABPβ and PGC-1α, the major transcriptional factor/cofactor for nuclear-encoded mitochondrial genes. Despite the reduced mitochondrial function in the liver, LSD1-LKO mice are protected from diet-induced hepatic steatosis and glucose intolerance, partially due to induction of hepatokine FGF21. Thus, LSD1 orchestrates a core regulatory network involving epigenetic modifications and NAD+ synthesis to control mitochondrial function and hepatokine production.

SUMMARY Elucidation of the sequence of events underlying the dynamic interaction between transcription factors and chromatin states is essential. Maternal transcription factors function at the top of the regulatory hierarchy to specify the primary germ layers at the onset of zygotic genome activation (ZGA). We focus on the formation of endoderm progenitor cells and examine the interactions between maternal transcription factors and chromatin state changes underlying the cell specification process. Endoderm-specific factors Otx1 and Vegt together with Foxh1 orchestrate endoderm formation by coordinated binding to select regulatory regions. These interactions occur before the deposition of enhancer histone marks around the regulatory regions, and these TFs recruit RNA polymerase II, regulate enhancer activity, and establish super-enhancers associated with important endodermal genes. Therefore, maternal transcription factors Otx1, Vegt, and Foxh1 combinatorially regulate the activity of super-enhancers, which in turn activate key lineage-specifying genes during ZGA., Graphical Abstract, In Brief How do maternal transcription factors interact with chromatin regions to coordinate the endodermal gene regulatory program? Paraiso et al. demonstrate that combinatorial binding of maternal Otx1, Vegt, and Foxh1 to select enhancers and super-enhancers in the genome controls endodermal cell fate specification during zygotic gene activation.

One of the first steps in cellular differentiation of vertebrate embryos is the formation of the three germ layers. Maternal pioneer transcription factors (TFs) bind to the regulatory regions of the embryonic genome prior to zygotic genome activation and initiate germ layer specification. While the involvement of maternal TFs in establishing epigenetic marks in whole embryos was addressed previously, how early pluripotent cells acquire spatially restricted epigenetic identity in embryos remain unknown. Here, we report that the H3K4me1 enhancer mark in each germ layer becomes distinct in germ layer specific regulatory regions, forming super-enhancers (SEs), by early gastrula stage. Distinct SEs are established in these germ layers near robustly regulated germ layer identity genes, suggesting that SEs are important for the canalization of development. Establishment of these enhancers requires a sequential function of maternal and zygotic TFs. By knocking down the expression of a critical set of maternal endodermal TFs, an overwhelming majority of the endodermal H3K4me1 marks are lost. Interestingly, this disappearance of endodermal marking coincides with the appearance of ectodermal and mesodermal H3K4me1 marks in the endoderm, suggesting a transformation in the chromatin state of these nuclei towards a more ecto-mesodermal state.De novomotif analysis to identify TFs responsible for the transformation recovers a profile for endodermal maternal TFs as well as their downstream target TFs. We demonstrate the importance of coordinated activities of maternal and zygotic TFs in defining a spatially resolved dynamic process of chromatin state establishment.

Carnitine palmitoyltransferase 1A (CPT1A) is a central player in lipid metabolism, catalyzing the first step to fatty acid oxidation (FAO). Inhibiting CPT1A, especially in the brain, can have several pharmacological benefits, such as in treating obesity and brain cancer. C75-CoA is a strong competitive inhibitor of CPT1A. However, due to its negatively charged nature, it has low cellular permeability. Herein, we report the use of poly-ion complex (PIC) micelles to deliver the specific CPT1A inhibitors (±)-, (+)-, and (-)-C75-CoA into U87MG glioma cells and GT1-7 neurons. PIC micelles were formed through charge-neutralization of the cargo with the cationic side chain of PEG-poly{N-[N'-(2-aminoethyl)-2-aminoethyl]aspartamide} (PEG-PAsp(DET)), forming particles with 55 to 65 nm diameter. Upon short-term incubation with cells, the micelle-encapsulated CPT1A inhibitors resulted in up to 5-fold reduction of ATP synthesis compared to the free drug, without an apparent decline in cell viability. Micelle treatment showed a discernible decrease in 14C-palmitate oxidation into CO2 and acid-soluble metabolites, confirming that the substantial lowering of ATP production has resulted from FAO inhibition. Micelle treatment also diminished IC50 by 2 to 4-fold over the free drug-treated U87MG after long-term incubation. To measure the cellular uptake of these CoA-adduct loaded PIC micelles, we synthesized a fluorescent CoA derivative and prepared Fluor-CoA micelles which showed efficient internalization in the cell lines, both in 2D and 3D culture models, especially in neurons where uptake reached up to 3-fold over the free dye. Our results starkly demonstrate that the PIC micelles are a promising delivery platform for anionic inhibitors of CPT1A in glioma cells and neurons, laying the groundwork for future research or clinical applications.

Lineage specification is governed by gene regulatory networks (GRNs) that integrate the activity of signaling effectors and transcription factors (TFs) on enhancers. Sox17 is a key transcriptional regulator of definitive endoderm development, and yet, its genomic targets remain largely uncharacterized. Here, using genomic approaches and epistasis experiments, we define the Sox17-governed endoderm GRN inXenopusgastrulae. We show that Sox17 functionally interacts with the canonical Wnt pathway to specify and pattern the endoderm while repressing alternative mesectoderm fates. Sox17 and β-catenin co-occupy hundreds of key enhancers. In some cases, Sox17 and β-catenin synergistically activate transcription apparently independent of Tcfs, whereas on other enhancers, Sox17 represses β-catenin/Tcf-mediated transcription to spatially restrict gene expression domains. Our findings establish Sox17 as a tissue-specific modifier of Wnt responses and point to a novel paradigm where genomic specificity of Wnt/β-catenin transcription is determined through functional interactions between lineage-specific Sox TFs and β-catenin/Tcf transcriptional complexes. Given the ubiquitous nature of Sox TFs and Wnt signaling, this mechanism has important implications across a diverse range of developmental and disease contexts.

Medical treatments of neuron-related disorders are limited due to the difficulty of targeting brain cells. Major drawbacks are the presence of the blood–brain barrier and the lack of specificity of the drugs for the diseased cells. Nanomedicine-based approaches provide promising opportunities for overcoming these limitations. Although many previous reviews are focused on brain targeting with nanomedicines in general, none of those are concerned explicitly on the neurons, while targeting neuronal cells in central nervous diseases is now one of the biggest challenges in nanomedicine and neuroscience. We review the most relevant advances in nanomedicine design and strategies for neuronal drug delivery that might successfully bridge the gap between laboratory and bedside treatment in neurology.

Summary Although Wnt/β-catenin signaling is generally conserved and well understood, the regulatory mechanisms controlling context-specific direct Wnt target gene expression in development and disease are still unclear. The onset of zygotic gene transcription in early embryogenesis represents an ideal, accessible experimental system to investigate context-specific direct Wnt target gene regulation. We combine transcriptomics using RNA-seq with genome-wide β-catenin association using ChIP-seq to identify stage-specific direct Wnt target genes. We propose coherent feedforward regulation involving two distinct classes of direct maternal Wnt target genes, which differ both in expression and persistence of β-catenin association. We discover that genomic β-catenin association overlaps with Foxh1-associated regulatory sequences and demonstrate that direct maternal Wnt target gene expression requires Foxh1 function and Nodal/Tgfβ signaling. Our results support a new paradigm for direct Wnt target gene co-regulation with context-specific mechanisms that will inform future studies of embryonic development and more widely stem cell-mediated homeostasis and human disease., Graphical Abstract, Highlights • Combining RNA-seq and β-catenin ChIP-seq identifies direct Wnt target genes • Two distinct classes of direct maternal Wnt/β-catenin target genes can be discerned • We propose coherent feedforward regulation of gene expression of the second class • Maternal Wnt target gene expression of both classes requires Nodal/Foxh1 signaling, Biological Sciences; Molecular Biology; Developmental Biology

For decades, the early development of the Xenopus embryo has been an essential model system to study the gene regulatory mechanisms that govern cellular specification. At the top of the hierarchy of gene regulatory networks, maternally deposited transcription factors initiate this process and regulate the expression of zygotic genes that give rise to three distinctive germ layer cell types (ectoderm, mesoderm, and endoderm), and subsequent generation of organ precursors. The onset of germ layer specification is also closely coupled with changes associated with chromatin modifications. This review will examine the timing of maternal transcription factors initiating the zygotic genome activation, the epigenetic landscape of embryonic chromatin, and the network structure that governs the process.

The crucial balance of stability in blood-circulation and tumor-specific delivery has been suggested as one of the challenges for effective bench-to-bedside translation of nanomedicines (NMs). Herein, we developed a supramolecularly enabled tumor-extracellular (Tex) pH-triggered NM that can maintain the micellar structure with the entrapped-drug during systemic circulation and progressively release drug in the tumor by rightly sensing heterogeneous tumor-pH. Desacetylvinblastine hydrazide (DAVBNH), a derivative of potent anticancer drug vinblastine, was conjugated to an aliphatic ketone-functionalized poly(ethylene glycol)-b-poly(amino acid) copolymer and the hydrolytic stability of the derived hydrazone bond was efficiently tailored by exploiting the compartmentalized structure of polymer micelle. We confirmed an effective and safe therapeutic application of Tex pH-sensitive DAVBNH-loaded micelle (Tex-micelle) in orthotopic glioblastoma (GBM) models, extending median survival to 1.4 times in GBM xenograft and 2.6 times in GBM syngeneic model, compared to that of the free DAVBNH. The work presented here offers novel chemical insights into the molecular design of smart NMs correctly sensing Tex-pH via programmed functionalities. The practical engineering strategy based on a clinically relevant NM platform, and the encouraging therapeutic application of Tex-micelle in GBM, one of the most lethal human cancers, thus suggests the potential clinical translation of this system against other types of common cancers, including GBM.

Lineage specification is governed by gene regulatory networks (GRNs) that integrate the activity of signaling effectors and transcription factors (TFs) on enhancers. Sox17 is a key transcriptional regulator of definitive endoderm development, and yet, its genomic targets remain largely uncharacterized. Here, using genomic approaches and epistasis experiments, we define the Sox17-governed endoderm GRN inXenopusgastrulae. We show that Sox17 functionally interacts with the canonical Wnt pathway to specify and pattern the endoderm while repressing alternative mesectoderm fates. Sox17 and β-catenin co-occupy hundreds of key enhancers. In some cases, Sox17 and β-catenin synergistically activate transcription independent of Tcfs, whereas on other enhancers, Sox17 represses ß-catenin/Tcf-mediated transcription to spatially restrict gene expression domains. Our findings establish Sox17 as a tissue-specific modifier of Wnt responses and point to a novel paradigm where genomic specificity of Wnt/ß-catenin transcription is determined through functional interactions between lineage-specific Sox TFs and ß-catenin/Tcf transcriptional complexes. Given the ubiquitous nature of Sox TFs and Wnt signaling, this mechanism has important implications across a diverse range of developmental and disease contexts.Key findingsSox17 regulates germ layer segregation by promoting endoderm differentiation while simultaneously repressing mesectoderm fates.Functional interactions between Sox17 and canonical Wnt-signaling is a major feature of the GRN controlling endoderm specification and patterning.Sox17 and β-catenin co-occupy a subset of enhancers to synergistically stimulate transcription in the absence of Tcfs.Sox17 regulates the spatial expression domains of Wnt/β-catenin-responsive transcription in the gastrula.

For decades, the early development of the Xenopus embryo has been an essential model system to study the gene regulatory mechanisms that govern cellular specification. At the top of the hierarchy of gene regulatory networks, maternally deposited transcription factors initiate this process and regulate the expression of zygotic genes that give rise to three distinctive germ layer cell types (ectoderm, mesoderm, and endoderm), and subsequent generation of organ precursors. The onset of germ layer specification is also closely coupled with changes associated with chromatin modifications. This review will examine the timing of maternal transcription factors initiating the zygotic genome activation, the epigenetic landscape of embryonic chromatin, and the network structure that governs the process.

Summary Background In 2013, the interim analysis of the Protocol for Herceptin as Adjuvant therapy with Reduced Exposure (PHARE) trial could not show that 6 months of adjuvant trastuzumab was non-inferior to 12 months. Here, we report the planned final analysis based on the prespecified number of occurring events. Methods PHARE is an open-label, phase 3, non-inferiority randomised trial of patients with HER2-positive early breast cancer comparing 6 months versus 12 months of trastuzumab treatment concomitant with or following standard neoadjuvant or adjuvant chemotherapy. The study was undertaken in 156 centres in France. Eligible patients were women aged 18 years or older with non-metastatic, operable, histologically confirmed adenocarcinoma of the breast and either positive axillary nodes or negative axillary nodes but a tumour of at least 10 mm. Participants must have received at least four cycles of a chemotherapy for this breast cancer and have started receiving adjuvant trastuzumab-treatment. Eligible patients were randomly assigned to either 6 months or 12 months of trastuzumab therapy duration between the third and sixth months of adjuvant trastuzumab. The randomisation was stratified by concomitant or sequential treatment with chemotherapy, oestrogen receptor status, and centre. The primary objective was non-inferiority in the intention-to-treat population in the 6-month group in terms of disease-free survival with a prespecified hazard margin of 1·15. This trial is registered with ClinicalTrials.gov, number NCT00381901. Findings 3384 patients were enrolled and randomly assigned to either 12 months (n=1691) or 6 months (n=1693) of adjuvant trastuzumab. One patient in the 12-month group and three patients in the 6-month group were excluded, so 1690 patients in each group were included in the intention-to-treat analysis. At a median follow-up of 7·5 years (IQR 5·3–8·8), 704 events relevant to disease-free survival were observed (345 [20·4%] in the 12-month group and 359 [21·2%] in the 6-month group). The adjusted hazard ratio for disease-free survival in the 12-month group versus the 6-month group was 1·08 (95% CI 0·93–1·25; p=0·39). The non-inferiority margin was included in the 95% CI. No differences in effects pertaining to trastuzumab duration were found in any of the subgroups. After the completion of trastuzumab treatment, rare adverse events occurred over time and the safety analysis remained similar to the previously published report. In particular, we found no change in the cardiac safety comparison, and only three additional cases in which the left ventricular ejection fraction decreased to less than 50% have been reported in the 12-month group. Interpretation The PHARE study did not show the non-inferiority of 6 months versus 12 months of adjuvant trastuzumab. Hence, adjuvant trastuzumab standard duration should remain 12 months. Funding The French National Cancer Institute.

It has recently been reported that a common side effect of translation-blocking morpholino antisense oligonucleotides is the induction of a set of innate immune response genes in Xenopus embryos and that splicing-blocking morpholinos lead to unexpected off-target mis-splicing events. Here, we present ananalysis of all publicly available Xenopus RNA sequencing (RNA-seq) data in a reexamination of the effects of translation-blocking morpholinos on the innate immune response. Our analysis does notsupport the authors' general conclusion, which wasbased on a limited number of RNA-seq datasets. Moreover, the strong induction of an immune response appears to be specific to the tbxt/tbxt2 morpholinos. The more comprehensive study presented here indicates that using morpholinos for targeted gene knockdowns remains of considerable value for the rapid identification of gene function.

Germ layer formation is among the earliest differentiation events in metazoan embryos. In triploblasts, three germ layers are formed, among which the endoderm gives rise to the epithelial lining of the gut tube and associated organs including the liver, pancreas and lungs. In frogs (Xenopus), where early germ layer formation has been studied intensively, the process of endoderm specification involves the interplay of dozens of transcription factors. Here, we review the interactions between these factors, summarized in a transcriptional gene regulatory network (GRN). We highlight regulatory connections conserved between Xenopus, zebrafish, mouse, and human endodermal lineages. Especially prominent is the conserved role and regulatory targets of the Nodal signaling pathway and the T-box transcription factors, Vegt and Eomes. Additionally, we highlight some network topologies and motifs, and speculate on their possible roles in development.

The use of gold nanorods (AuNRs) that produce heat in response to near infrared (NIR) irradiation is an attractive approach to cancer photothermal therapy. AuNRs are usually prepared by using a highly toxic detergent: cetyltrimethylammonium bromide (CTAB). Thus, the removal of CTAB from the reaction mixture, and further stabilization of the surface of the AuNRs is required. In the present study, AuNRs were encapsulated in a multifunctional envelope-type nano device (AuNR-MEND) formed with an SS-cleavable and pH-activated lipid-like material. In the process of encapsulation, AuNRs were first stabilized with bovine serum albumin (AuNR-BSA), and then further encapsulated in the lipid envelope by the ethanol dilution method. The in vitro photothermal cytotoxicity of AuNR-MEND was further demonstrated on 4T1 breast cancer cells. After NIR radiation, the temperature of the medium was increased to approximately 60°C, and cell viability was drastically decreased to approximately 11%. However, this cytotoxic effect cannot simply be explained by medium heating. It therefore appears that intracellular delivery of the AuNRs is a key factor for achieving a high degree of cytotoxicity. Dose dependent cytotoxicity data revealed that a higher dose of AuNR-MEND resulted in the complete destruction of the cells when they were subjected to NIR irradiation, while the cell survival rate reached a plateau at 30% in the case of AuNR-BSA. Apoptosis was induced after treatment with the nanoparticles. AuNR-MEND showed superior cellular uptake activity over AuNR-BSA. Thus, delivering AuNR by means of functionalized lipid nanoparticles represents a promising approach to induce NIR-triggered apoptosis.

The interplay between transcription factors and chromatin dictates gene regulatory network activity. Germ layer specification is tightly coupled with zygotic gene activation and, in most metazoans, is dependent upon maternal factors. We explore the dynamic genome-wide interactions of Foxh1, a maternal transcription factor that mediates Nodal/TGF-β signaling, with cis-regulatory modules (CRMs) during mesendodermal specification. Foxh1 marks CRMs during cleavage stages and recruits the co-repressor Tle/Groucho in the early blastula. We highlight a population of CRMs that are continuously occupied byFoxh1 and show that they are marked by H3K4me1, Ep300, and Fox/Sox/Smad motifs, suggesting interplay between these factors in gene regulation. We also propose a molecular "hand-off" between maternal Foxh1 and zygotic Foxa at these CRMs to maintain enhancer activation. Our findings suggest that Foxh1 functions at the top of a hierarchy of interactions by marking developmental genes for activation, beginning with the onset of zygotic gene expression.

Metastatic breast cancer chemotherapy in the elderly is considered effective in carefully selected patients, but there is little data regarding its effect in vulnerable patients.We evaluated tumour response (primary endpoint), feasibility and outcomes after six courses of an adapted dose of pegylated liposomal doxorubicin (PLD) (40 mg/m(2) every 28 days) as first-line chemotherapy for hormone-resistant MBC.Of 60 patients70 years (median 77 years), 15% had performance status ≥2 and 73% had visceral metastases. Geriatric assessment included: ≥2 comorbidities, 42%; ≥1 deficiency in Activities of Daily Living (ADL), 10% and Instrumental ADL (IADL), 82%; living in residential homes, 12%; albumin35 g/L, 17%; body mass index (BMI)21, 20%; depression, 17%; and lymphocytes ≤1 × 10(3)/mm(3), 27%. Complete response, partial response and stable disease were observed in 5%, 15% and 60%, respectively, but only 48% completed six cycles. Treatment discontinuations were mostly due to disease progression (18%) and non-haematological (NH) toxicities (22%). Eight patients died during treatment (three possibly related to PLD), and 15 had unplanned hospital admissions. Exploratory analyses to identify geriatric covariates associated with treatment outcomes revealed severe haematological toxicities significantly correlated with lymphocytes ≤1 × 10(3)/mm(3). NH toxicities correlated with age ≥80 years and living in residential homes. Progression-free survival (median 6.1 months) decreased with age, deficiency in IADL, cardiac dysfunction and living in residential homes. Overall survival (median 15.7 months) also decreased with living in residential homes.Despite manageable haematological toxicities and expected response rates, PLD feasibility was poor in unselected elderly patients.

Gene regulatory networks (GRNs) involve highly combinatorial interactions between transcription factors and short sequence motifs in cis-regulatory modules of target genes to control cellular phenotypes. The GRNs specifying most cell types are largely unknown and are the subject of wide interest. A catalog of transcription factors is a valuable tool toward obtaining a deeper understanding of the role of these critical effectors in any biological setting. Here we present a comprehensive catalog of the transcription factors for the diploid frog Xenopus tropicalis. We identify 1235 genes encoding DNA-binding transcription factors, comparable to the numbers found in typical mammalian species. In detail, the repertoire of X. tropicalis transcription factor genes is nearly identical to human and mouse, with the exception of zinc finger family members, and a small number of species/lineage-specific gene duplications and losses relative to the mammalian repertoires. We applied this resource to the identification of transcription factors differentially expressed in the early gastrula stage embryo. We find transcription factor enrichment in Spemann's organizer, the ventral mesoderm, ectoderm and endoderm, and report 218 TFs that show regionalized expression patterns at this stage. Many of these have not been previously reported as expressed in the early embryo, suggesting thus far unappreciated roles for many transcription factors in the GRNs regulating early development. We expect our transcription factor catalog will facilitate myriad studies using Xenopus as a model system to understand basic biology and human disease.

Advances in RNA sequencing technologies have led to the surprising discovery that a vast number of transcripts emanate from regions of the genome that are not part of coding genes. Although some of the smaller ncRNAs such as microRNAs have well-characterized functions, the majority of long ncRNA (lncRNA) functions remain poorly understood. Understanding the significance of lncRNAs is an important challenge facing biology today. A powerful approach to uncovering the function of lncRNAs is to explore temporal and spatial expression profiling. This may be particularly useful for classes of lncRNAs that have developmentally important roles as the expression of such lncRNAs will be expected to be both spatially and temporally regulated during development. Here, we take advantage of our ultra-high frequency (temporal) sampling of Xenopus embryos to analyze gene expression trajectories of lncRNA transcripts over the first 3 days of development. We computationally identify 5,689 potential single- and multi-exon lncRNAs. These lncRNAs demonstrate clear dynamic expression patterns. A subset of them displays highly correlative temporal expression profiles with respect to those of the neighboring genes. We also identified spatially localized lncRNAs in the gastrula stage embryo. These results suggest that lncRNAs are involved in regulatory roles during early embryonic development.

Purpose To define the recommended dose of the association of weekly irinotecan (Iri) and cisplatin (CP) with pelvic radiotherapy in Locally Advanced Cervical Cancer. Patients and methods Stage IB2-IV cervix cancer patients were treated with escalating doses of Iri starting from 30 mg/m 2 and a fixed dose of CP at 20 mg/m 2 , both weekly concomitantly with a 45-Gy pelvic irradiation. Results Fifteen patients entered the study, 6 at level 1 (Iri 30 mg/m 2 ), 3 at level 2 (Iri 40 mg/m 2 ) and 6 at intermediate dose (Iri 35 mg/m 2 ). Median age was 47 years (34-72), FIGO stage IB ( n =1), IIB ( n =7), III ( n =6), IVA ( n =1). The recommended dose was weekly Iri 35 mg/m 2 and CP 20 mg/m 2 . Dose limiting toxicities (grades 3–4) were diarrhea, abdominal pain, febrile neutropenia and fatigue. Conclusion In cervix cancer patients, radiosensitization with weekly cisplatin and irinotecan is feasible, and the recommended doses are cisplatin 20 mg/m 2 /week and irinotecan 35 mg/m 2 /week for future phase II studies.

Advanced ovarian carcinoma in early progression (6 months) (AOCEP) is considered resistant to most cytotoxic drugs. Gemcitabine (GE) and oxaliplatin (OXA) have shown single-agent activity in relapsed ovarian cancer. Their combination was tested in patients with AOCEP in phase II study. Fifty patients pre-treated with platinum-taxane received q3w administration of OXA (100 mg m(-2), d1) and GE (1000 mg m(-2), d1, d8, 100-min infusion). Patient characteristics were a : median age 64 years (range 46-79),and 1 (84%) or 2 (16%) earlier lines of treatment. Haematological toxicity included grade 3-4 neutropaenia (33%), anaemia (8%), and thrombocytopaenia (19%). Febrile neutropaenia occurred in 3%. Non-haematological toxicity included grade 2-3 nausea or vomiting (34%), grade 3 fatigue (25%),and grade 2 alopecia (24%). Eighteen (37%) patients experienced response. Median progression-free (PF) and overall survivals (OS) were 4.6 and 11.4 months, respectively. The OXA-GE combination has high activity and acceptable toxicity in AOCEP patients. A comparison of the doublet OXA-GE with single-agent treatment is warranted.

Background Variable rates of HER2 protein overexpression and gene amplification have been reported in advanced ovarian cancers (AOC). Trastuzumab, tested only as a single agent, has been shown to achieve 7% response in heavily pretreated patients with AOC with 3+ and 2+ HER2 immunostaining by immunohistochemistry (IHC). The GINECO trial explored the combination of trastuzumab with paclitaxel and carboplatin in patients with resistant AOC (< 6 months) and HER2 gene amplification.

The objective of the study is to determine whether surgery influences the outcome of stage IV ovarian cancer. The study design is as follows: From May 1995 to December 2000, 129 patients with FIGO stage IV ovarian cancer, recruited in 42 centers, were prospectively included in GINECO first-line randomized studies of platinum-based regimens with paclitaxel administered simultaneously or sequentially. In all, 109 were eligible for this study. Standard peritoneal cytoreductive surgery was defined as a procedure including at least total hysterectomy, bilateral salpingo-oophorectomy, omentectomy, and peritoneal debulking. Surgery was considered optimal if residual lesions were smaller than 1 cm. The Kaplan-Meier method was used to compare survival. Initial abdominopelvic cytoreductive surgery was considered standard in 55 (54%) patients. Abdominopelvic surgery was optimal in 29 patients and nonoptimal in 26. Twenty-two (22%) patients had a simple biopsy, and 25 (24%) patients underwent substandard surgery. Twenty-two of these 47 patients without initial standard surgery underwent a second surgical procedure, and 17 of the 22 patients completed standard surgery. The median overall survival time in the entire population was 24.3 months (95% confidence interval [CI], 19.5-29.1 months). Patients treated without a cytoreductive surgical procedure had significantly worse median survival (15.1 months; 95% CI, 5.4-24.9 months) than patients who had optimal primary surgery (22.9 months; 95% CI, 15.6-30.1 months), nonoptimal primary surgery (27.1 months; 95% CI, 21.2-32.9 months), or neoadjuvant chemotherapy followed by surgery (45.5 months; 95% CI, 23.5-67.5 months) (P= .001). In conclusion, this study shows a significant benefit of debulking surgery in stage IV ovarian cancer patients who responded to neoadjuvant chemotherapy. Neoadjuvant chemotherapy can help to select patients for surgery.

Background: Patients with recurrent/metastatic endometrial cancer that progresses after chemotherapy have limited treatment options and poor outcomes. Preclinical data suggest the oral mammalian target of rapamycin inhibitor everolimus may provide clinical benefit in these patients. Methods: In this multicenter, open-label, phase 2 study, patients with advanced or metastatic endometrial cancer refractory to one or two previous chemotherapy regimens received everolimus 10 mg per day until progression or unacceptable toxicity. Primary end point was the non-progressive disease rate at 3 months. Secondary end points included duration of response, progression-free, and overall survival (OS), and safety. Results: Forty-four patients were enrolled (median age, 65 years); 66% received one previous chemotherapy regimen. The 3-month non-progressive disease rate was 36% (95% confidence interval 22–52%), including two patients (5%) with partial response (PR). At 6 months, two additional patients experienced PR. Median duration of response was 3.1 months. Median progression-free and OS were 2.8 months and 8.1 months, respectively. The most common adverse events were anaemia (100%), fatigue (93%), hypercholesterolaemia (81%), and lymphopenia (81%). Conclusion: Everolimus demonstrated efficacy and acceptable tolerability in patients with chemotherapy-refractory advanced or metastatic endometrial cancer. These results support the further development of phosphatidylinositol 3-kinase-targeted therapies in endometrial cancer.

Fotemustine and dacarbazine constitute the most active single chemotherapeutic agents in the treatment of melanoma. In this phase II study we evaluated the activity and toxicity of a combination of fotemustine, dacarbazine and vindesine as a means of increasing response rate and survival time. Between September 1989 and November 1993, 43 patients with advanced melanoma were treated with a combination of 100 mg/m2 fotemustine on days 1 and 8, 250 mg/m2 dacarbazine on days 15 and 16 and 2 mg/m2 vindesine on days 15 and 16 as induction treatment. After a 5-week rest period, the patients exhibiting a response or stable disease received the same drugs administered once every 28 days as maintenance therapy until either progression or toxicity was observed. Among 41 evaluable patients, there were six complete responses and eight partial responses. The overall response rate was 32% (95% confidence interval: 18-46%), with 8 months median duration of response. Median survival time was 10 months. This regimen was well tolerated. From this large phase II study, we conclude that such a combination is active against advanced malignant melanoma and seems to be more effective than fotemustine or dacarbazine used alone, especially on visceral metastatic sites.

Background Platinum rechallenge or weekly topotecan in combination have not been evaluated in randomized trials for resistant recurrent ovarian cancer (ROC). Methods Patients with ROC after first- or second-line treatment including a platinum and taxane and progression within 6 months were randomized to weekly paclitaxel (wP, 80 mg/m2/week) alone or in combination with carboplatin (C, area under the curve of 5 mg/ml/min every 4 weeks) or weekly topotecan (wT, 3 mg/m2/week). Primary end point was progression-free survival (PFS) comparing wP and combination therapy. Results Patients (n = 165) received a median three cycles in each arm. Nonhematologic toxicity was not different, except increased hypersensitivity reactions with wP + C. Grade 3–4 hematologic toxic effects with wP, wP + C, and wP + wT, respectively, were neutropenia in 13%, 54%, and 42%; febrile neutropenia in 0%, 4%, and 5%; and anemia in 6%, 19%, and 29%. Response rates were 35%, 37%, and 39%, and median PFS times were 3.7, 4.8, and 5.4 months, respectively. PFS was not significantly different among the treatment arms [hazard ratio (HR) 0.922; 95% confidence interval (CI) 0.765–1.111; P = 0.46] or between monotherapy and combination therapy (HR 0.951; 95% CI 0.686–1.318; P = 0.76). Conclusions Combination chemotherapy in platinum-resistant ROC was more toxic than weekly paclitaxel and did not significantly prolong PFS.

Background This phase II study evaluated the clinical benefit of pegylated liposomal doxorubicin (PLD) and docetaxel (Taxotere) as first-line therapy for metastatic breast cancer (MBC). Patients and methods MBC patients were enrolled to receive six cycles of PLD 35 mg/m2 (day 1) and docetaxel 40 mg/m2 (days 1 and 15), every 28 days (group A). Because of unacceptable toxic effects, doses were modified to PLD 30 mg/m2 (day 1) and docetaxel 75 mg/m2 (day 2), every 3 weeks (group B). The primary end point was clinical benefit. Results Sixty-seven patients were included (group A, 53; group B, 14). In both groups, the median number of cycles delivered was 4 and the overall dose intensity was 82% for docetaxel and 71% for PLD. In group A, main toxic effects were hematologic, palmar–plantar erythrodysesthesia (PPE), and stomatitis. In group B, higher rates of grade 3–4 PPE, febrile neutropenia, and hematologic toxic effects were reported. The rate of clinical benefit was 47%. Among patients with a measurable disease, 49% achieved a partial response, 27% had a stable disease, and 13% progressed, according to RECIST criteria. Conclusion The combination of PLD and docetaxel delivered at planned doses in this study yields unacceptable toxicity and should not be used routinely in patients with MBC.

Requirements for reduction in the duration of short circuit faults on transmission lines are based on significant system congestion and increased numbers of customers with loads sensitive to voltage variations. High-speed fault clearing for different faults on transmission lines can be achieved by advanced communications based protection schemes. However, they have the disadvantage that they require the availability of communications between the two ends of the line, which in many cases are not available. Significant improvement in the performance of non-communications based protection solutions can be made by the implementation of adaptive protection at the transmission level of the system. The grounding of transformers and their state, state of generators, the configuration of the system and the dynamic behavior of the system are analyzed from the point of view of the performance of transmission line protection relays. Special attention is paid to the cases of parallel, double circuit or multi-terminal lines. The effect of line state and grounding is analyzed. The performance of relays under dynamic loading conditions and power swings is later described in order to discuss the challenges of detecting of faults occurring under such conditions. After it is clear what are the conditions that have effect on the performance of non-communications based transmission line protection relays, the paper describes the methods for detection of changes in the different factors that affect the operation of the relays. Features in the transmission line protection relays that allow them to adapt to the changes in the system condition are described later in the paper. Monitoring of the state of breakers or switches, interface with the substation automation system, detection of faults under dynamic system conditions, changing of setting groups or use of the programmable scheme logic are discussed. The performance of the different adaptive functions used in transmission line protection relays need to be tested to ensure their correct operation during the changing system conditions. Tools for testing of these functions are discussed at the end of the paper.

Objective cisplatin (Cp) plus topotecan (Tc) is the first combination chemotherapy to demonstrate a survival advantage over cisplatin alone in advanced cervical cancer. Combining Cp and Tc with an epidermal growth factor receptor (EGFR) inhibitor such as cetuximab (Ce) may increase the activity of chemotherapy. Methods Patients with advanced cervical squamous cell cancer or adenocarcinoma and at least one measurable target received intravenous Cp 50 mg/m 2 on day 1 plus Tc 0.75 mg/m 2 /day from days 1 to 3 every 3 weeks combined with Ce (initial dose of 400 mg/m 2 followed by subsequent weekly dose of 250 mg/m 2 ). Objective response rate according to RECIST criteria was the primary end point; safety, progression free survival (PFS) and overall survival (OS) were secondary end points. Results Between April and July 2007, 19 out of the 44 planned patients were accrued before the study was stopped early due to excessive toxicity. The most frequent adverse event was severe myelosuppression with grades 3–4 neutropenia (72%), grades 3–4 thrombocytopenia (61%), and grade 3 anemia (44.5%). The main grades 3–4 non-hematologic toxicities were infection (39%) and febrile neutropenia (28%), skin reactions (22%), renal toxicity (11%), and pulmonary embolism (11%). Five (28%) patients died during the treatment including 3 deaths related to treatment toxicity. Six (32%) evaluable patients achieved a partial response. The median times of PFS and OS were 172 and 220 days, respectively. Conclusion In this phase II trial, the combination Cp–Tc–Ce induced a high rate of serious adverse and/or fatal events at standard dose and schedule. Cetuximab plus platinum-based combination chemotherapy should be further explored with caution in the future in advanced cervix cancer.

Background: Platinum-based chemotherapy is standard second-line treatment of patients with advanced ovarian cancer (AOC) in late relapse. Pegylated liposomal doxorubicin (PLD) has significant single-agent activity in this setting. Therefore, we evaluated the use of PLD plus carboplatin in this patient population. Patients and methods: PLD 30 mg/m2 followed by carboplatin at area under the curve (AUC) 5 mg·min/ml, repeated every 28 days for a maximum of nine cycles, was administered to 104 women with AOC relapsing ≥6 months after completion of first- or second-line therapy with platinum-taxane-based regimens. Results: Overall response was 63%, with a 38% complete response, median progression-free survival of 9.4 months, and median overall survival (OS) of 32 months. Grade 3 or 4 neutropenia occurred in 51% of patients, but febrile neutropenia in only 3%. Nonhematologic toxic effects were primarily grades 1 and 2, with low rates of alopecia and neurotoxicity. Conclusions: PLD plus carboplatin is highly effective, prolongs OS, and is well tolerated in women with AOC in late relapse previously treated with both platinum and taxanes. Evaluation of this regimen in phase III trials is warranted.

In this paper, analysis of the phase and neutral current harmonics in a three-phase computer power system are carried out under distorted and/or unbalanced supply voltage conditions. Furthermore, the impact of background voltage harmonics on the resultant harmonic currents produced by a large concentration of personal computers is investigated. Results obtained from the proposed approach are compared to simulation results obtained using the PSPICE software package

In this paper, analysis of the phase and neutral current harmonics in a three-phase computer power system is carried out under distorted and/or unbalanced supply voltage conditions. The impact of background voltage harmonics on the resultant harmonic currents produced by a large concentration of personal computers is investigated. Results obtained from the proposed approach, with Matlab, are compared to those obtained from simulations using the PSPICE software package.

Background Despite numerous studies, no biological marker has been identified that accurately predicts prognosis of advanced ovarian cancer. Tumors from a homogeneous population of 117 patients with a stage III/IV ovarian cancer, enrolled in a multicenter prospective GINECO clinical trial were analyzed retrospectively. Patients and methods All patients received the same platinum-based combination therapy and were followed-up for a median of 68 months. Tumor expression of Ki67, BCL-2, BAX, P53 or c-erbB-2 proteins was evaluated immunohistochemically on paraffin-embedded tissues and their prognostic impact analyzed. Results The median rate of Ki67-positive nuclear area was 30%. BCL-2, BAX and P53 proteins were expressed in 52, 54 and 71% of the tumors, respectively, while HER-2 protein was overexpressed in 16%. Only HER-2 overexpression was significantly associated with shorter progression-free survival and overall survival. According to our multivariate analysis, the HER-2 prognostic impact was independent of classical clinical prognostic factors. Conclusion HER-2 appeared to influence the outcome of advanced ovarian cancer patients included in a clinical trial with prolonged follow-up, thereby suggesting that HER-2 is a potential target for treatment of this cancer.

A multicenter phase II study to evaluate the antitumor effect and safety of docetaxel in combination with cisplatin as first-line chemotherapy for advanced ovarian cancer.Enrolled in the study were 45 patients who were to receive six courses of docetaxel 75 mg/m(2) plus cisplatin 75 mg/m(2) every 21 days with hydration and steroid prophylaxis after initial debulking surgery. Imaging techniques and radiography were used to assess clinical tumor response, and second-look surgery was required for patients with complete clinical responses and for those without clinically measurable disease.The overall clinical response rate in 29 patients with clinically measurable disease was 58% (41% complete response). A complete pathologic response was seen in 9 of 34 patients who underwent second-look laparotomy, while microscopic disease was found in 10 patients. The median time to progression was 14.4 months (95% CI 8.4-20.4 months), with a median overall survival of 43 months (95% CI 21.1-65.0 months). Patients received a median number of six cycles at a dose intensity of 98%. Grade 3-4 neutropenia was seen in 80% of patients, but was manageable. No patients withdrew because of fluid retention.The combination of docetaxel with cisplatin confers high clinical and pathologically verified tumor response rates and is well tolerated in the first-line management of advanced ovarian cancer.

This is a relatively common situation since metastases of indeterminate origin represent 5 to 10% of all cancers, depending on the series and the definition adopted. The most restrictive definition only includes metastases whose origin cannot be determined even at autopsy [7]. A more useful definition includes all patients in whom a primary tumor is not demonstrated despite clinical and intensive radiological investigation at the time when the therapeutic decision had to be taken [3]. This latter definition is more commonly used.

Adenocarcinoma of the kidney is the commonest form of primary renal tumor in the adult. Early diagnosis is possible for this tumor due to its characteristic mode of spread and generally slow growth rate. In a third of cases, the diagnosis is made incidentally when the patient is still asymptomatic. In the metastatic phase, the presenting signs are variable depending on which organs are involved, and will guide the choice of investigations.

The clinical activity and toxicity of the triple combination of paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ), cyclophosphamide, and cisplatin was assessed in both previously treated and untreated women with advanced ovarian carcinoma. Paclitaxel 175 mg/m2 was administered over 3 hours following standard premedication (prednisolone, dexchlorpheniramine, and cimetidine). Cisplatin 80 mg/m2 and cyclophosphamide 600 mg/m2 were given 6 to 12 hours after paclitaxel. Treatment was given at 3-week intervals for six cycles. Twenty-seven patients entered the study; 23 were evaluable for toxicity and 17 for response. Paclitaxel appeared to add additional efficacy to the standard cisplatin/cyclophosphamide regimen. Both the overall and complete remission rates were very high (88% and 70%, respectively), and histologically confirmed complete remissions exceeded 60%. Longer follow-up is needed to determine the duration of these responses. The primary toxicities included leukoneutropenia, peripheral neuropathy, asthenia, and alopecia. Only two of 23 patients withdrew because of toxicity, however, and only two treatment cycles were complicated by neutropenic fever requiring intravenous antibiotics. No life-threatening toxicities were encountered, although the peripheral neuropathy was poorly and slowly reversible and may have a significant impact on the patients' quality of life.

In this phase I/II study, we assessed the impact of paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ) in the treatment of advanced ovarian carcinoma combined with the standard regimen cisplatin/cyclophosphamide given as follows: paclitaxel 175 mg/m2 (over 3 hours perfusion with standard premedication), cisplatin 80 mg/m2 (6 to 12 hours after paclitaxel), and cyclophosphamide 400 mg/m2. From February 1994 to January 1996, 27 patients (median age, 55 years; age range, 35 to 74 years) were entered into the study. Eight patients had distant metastases and 19 had early locoregional disease (stage III, 18 patients; stage IC, one patient). Twenty-two patients had undergone prior surgery (simple biopsy, six patients; optimally debulked, nine patients; suboptimally debulked, seven patients). Twenty-one patients had received no prior chemotherapy and six were previously treated with at least one platinum-based regimen. A maximum of six courses of paclitaxel/cisplatin/cyclophosphamide were given every 21 days. Twenty-three patients were evaluable for toxicity: neutropenia (World Health Organization grade 3/4), 91% of patients; thrombopenia (World Health Organization grade 3/4), 13% of patients; two episodes of neutropenia with fever; and neurotoxicity grade 3, 17% of patients. Alopecia grade 3 was reported in all patients. No hypersensitivity reactions and no cardiac toxicity was observed. Among 17 patients evaluable for response (patients with stage IV disease or stage III suboptimally debulked), 12 (70%) clinical complete responses (CRs) and three (18%) partial responses were observed. Among the 12 patients with CRs, 10 underwent second-look laparotomy and seven of them (70%) achieved a pathologic CR. In the group of 11 chemotherapy-naive patients evaluable for response, eight (72%) achieved a CR and three (28%) achieved a partial response. This combination seems to be safe, with very acceptable toxicity, and also seems to be highly active in the treatment of patients with advanced ovarian carcinoma.

Endometrial carcinoma is one of the most common gynaecological cancers in Western countries. About 75% of the patients present limited disease, confined to the uterus that can be cured by surgery. However, one third of the patients will need systemic treatment because of metastatic or relapsing disease. Hormonotherapy response rates are less than 20%. In monochemotherapy, the higher response rates are constantly observed with doxorubicin or cisplatinum (25-35%). Most commonly used combination are CAP (cyclophosphamide, doxorubicin, cisplatinum) or AP (doxorubicin, cisplatinum), giving 35 to 60% of objective responses. Recent results of large randomized trials have demonstrated marginal, if any, effect of cyclophosphamide and superiority of doxorubicin-cisplatinum combination compared to doxorubicin alone for response and survival. Chemotherapy as hormonotherapy remains palliative. Median response duration is 4 to 6 months and median overall survival duration is 7 to 10 months. Currently, hormonotherapy-chemotherapy combination have not been proved to be more effective than chemotherapy alone.

Thirty-three patients, with histologically proven advanced nonsmall cell lung cancer (NSCLC) were treated at Institut Curie with a three-drug combination chemotherapy. The regimen consisted of cisplatin 25 mg/m2 per day continuous infusion (CI) and 5-fluorouracil (5-FU) CI 600 mg/m2 on 5 consecutive days with 30 minutes infusion navelbine 25 mg/m2 on days 1 and 5 only. Cycles were repeated every 28 days. Response was evaluated after three cycles. One patient died of an ischemic cerebrovascular stroke after the third cycle. Thirty-two pts were evaluable. Partial response was achieved in 11/20 patients with stage III disease (55%) and in 7/12 patients with stage IV disease (58%). WHO grade 4 toxicities were leucopenia 31%, thrombocytopenia 4%, and mucositis 4%. The tolerance was acceptable. Median survival was 19 months for stage III and 9.5 months for stage IV. We conclude that the combination of 5-FU, navelbine, and cisplatin is an effective chemotherapy regimen for patients with advanced stage NSCLC.