Governing Ecological Connectivity in Cross-Scale Dependent Systems.

Ecosystem management and governance of cross-scale dependent systems require integrating knowledge about ecological connectivity in its multiple forms and scales. Although scientists, managers, and policymakers are increasingly recognizing the importance of connectivity, governmental organizations may not be currently equipped to manage ecosystems with strong cross-boundary dependencies. Managing the different aspects of connectivity requires building social connectivity to increase the flow of information, as well as the capacity to coordinate planning, funding, and actions among both formal and informal governance bodies. We use estuaries in particular the San Francisco Estuary, in California, in the United States, as examples of cross-scale dependent systems affected by many intertwined aspects of connectivity. We describe the different types of estuarine connectivity observed in both natural and human-affected states and discuss the human dimensions of restoring beneficial physical and ecological processes. Finally, we provide recommendations for policy, practice, and research on how to restore functional connectivity to estuaries.

The Biological Deserts Fallacy: Cities in Their Landscapes Contribute More than We Think to Regional Biodiversity.

Cities are both embedded within and ecologically linked to their surrounding landscapes. Although urbanization poses a substantial threat to biodiversity, cities also support many species, some of which have larger populations, faster growth rates, and higher productivity in cities than outside of them. Despite this fact, surprisingly little attention has been paid to the potentially beneficial links between cities and their surroundings. We identify five pathways by which cities can benefit regional ecosystems by releasing species from threats in the larger landscape, increasing regional habitat heterogeneity and genetic diversity, acting as migratory stopovers, preadapting species to climate change, and enhancing public engagement and environmental stewardship. Increasing recognition of these pathways could help cities identify effective strategies for supporting regional biodiversity conservation and could provide a science-based platform for incorporating biodiversity alongside other urban greening goals.

Letrozole and palbociclib versus chemotherapy as neoadjuvant therapy of high-risk luminal breast cancer.

Background: Palbociclib is a CDK4/6 inhibitor with demonstrated efficacy and safety in combination with endocrine therapy in advanced luminal breast cancer (LBC). We evaluated the respective efficacy and safety of chemotherapy and letrozole-palbociclib (LETPAL) combination as neoadjuvant treatment in patients with high-risk LBC. Patients and methods: NeoPAL (UCBG10/4, NCT02400567) is a randomised, parallel, non-comparative phase II study. Patients with ER-positive, HER2-negative, Prosigna®-defined luminal B, or luminal A and node-positive, stage II-III breast cancer, not candidate for breast-conserving surgery, were randomly assigned to either letrozole (2.5 mg daily) and palbociclib (125 mg daily, 3 weeks/4) during 19 weeks, or to FEC100 (5FU 500 mg/m2, epirubicin 100 mg/m2, cyclophosphamide 500 mg/m2)×3 21-day courses followed by docetaxel 100 mg/m2×3 21-day courses. Primary end point was residual cancer burden (RCB 0-I rate). Secondary end points included clinical response, proliferation-based markers, and safety. Results: Overall, 106 patients were randomised [median Prosigna® ROR Score 71 (22-93)]. RCB 0-I was observed in four and eight patients in LETPAL [7.7% (95% CI 0.4-14.9)] and chemotherapy [15.7% (95% CI 5.7-25.7)] arms, respectively. Pathological complete response rates were 3.8% and 5.9%. Clinical response (75%) and breast-conserving surgery rates (69%) were similar in both arms. Preoperative Endocrine Prognostic Index 0 scores (breast cancer-specific survival) were observed in 17.6% and 8.0% of patients in LETPAL and chemotherapy arms, respectively. Safety profile was as expected, with 2 versus 17 serious adverse events (including 11 grade 4 serious AEs in the chemotherapy arm). Conclusion: LETPAL combination was associated with poor pathological response but encouraging clinical and biomarker responses in Prosigna®-defined high-risk LBC. Contemporary chemotherapy regimen was associated with poor pathological and biomarker responses, with a much less favourable safety profile. LETPAL combination might represent an alternative to chemotherapy in early high-risk LBC. Clinical Trial Number: NCT02400567.

A phase II trial of abiraterone acetate plus prednisone in patients with triple-negative androgen receptor positive locally advanced or metastatic breast cancer (UCBG 12-1).

BACKGROUND: Several expression array studies identified molecular apocrine breast cancer (BC) as a subtype that expresses androgen receptor (AR) but not estrogen receptor α. We carried out a multicentre single-arm phase II trial in women with AR-positive, estrogen, progesterone receptor and HER2-negative (triple-negative) metastatic or inoperable locally advanced BC to assess the efficacy and safety of abiraterone acetate (AA) plus prednisone. PATIENTS AND METHODS: Patients with a metastatic or locally advanced, centrally reviewed, triple-negative and AR-positive (≥10% by immunohistochemistry, IHC) BC were eligible. Any number of previous lines of chemotherapy was allowed. AA (1000 mg) was administered once a day with prednisone (5 mg) twice a day until disease progression or intolerance. The primary end point was clinical benefit rate (CBR) at 6 months defined as the proportion of patients presenting a complete response (CR), partial response (PR) or stable disease (SD) ≥6 months. Secondary end points were objective response rate (ORR), progression-free survival (PFS) and safety. RESULTS: One hundred and forty-six patients from 27 centres consented for IHC central review. Of the 138 patients with sufficient tissue available, 53 (37.6%) were AR-positive and triple-negative, and 34 of them were included from July 2013 to December 2014. Thirty patients were eligible and evaluable for the primary end point. The 6-month CBR was 20.0% [95% confidence interval (CI) 7.7%-38.6%], including 1 CR and 5 SD ≥6 months, 5 of them still being under treatment at the time of analysis (6.4+, 9.2+, 14.5+, 17.6+, 23.4+ months). The ORR was 6.7% (95% CI 0.8%-22.1%). The median PFS was 2.8 months (95% CI 1.7%-5.4%). Fatigue, hypertension, hypokalaemia and nausea were the most common drug-related adverse events; the majority of them being grade 1 or 2. CONCLUSIONS: AA plus prednisone treatment is beneficial for some patients with molecular apocrine tumours and five patients are still on treatment. CLINICALTRIALSGOV: NCT01842321.

Prognostic value of EndoPredict test in patients with hormone receptor-positive, human epidermal growth factor receptor 2-negative primary breast cancer screened for the randomized, double-blind, phase III UNIRAD trial.

BACKGROUND: The purpose of this study was to evaluate the prognostic value of the multigene EndoPredict test in prospectively collected data of patients screened for the randomized, double-blind, phase III UNIRAD trial, which evaluated the addition of everolimus to adjuvant endocrine therapy in high-risk, hormone receptor-positive, human epidermal growth factor receptor 2 (HER2)-negative early breast cancer. PATIENTS AND METHODS: Patients were classified into low or high risk according to the EPclin score, consisting of a 12-gene molecular score combined with tumor size and nodal status. Association of the EPclin score with disease-free survival (DFS) and distant metastasis-free survival (DMFS) was evaluated using Kaplan-Meier estimates. The independent prognostic added value of EPclin score was tested in a multivariate Cox model after adjusting on tumor characteristics. RESULTS: EndoPredict test results were available for 768 patients: 663 patients classified as EPclin high risk (EPCH) and 105 patients as EPclin low risk (EPCL). Median follow-up was 70 months (range 1-172 months). For the 429 EPCH randomized patients, there was no significant difference in DFS between treatment arms. The 60-month relapse rate for patients in the EPCL and EPCH groups was 0% and 7%, respectively. Hazard ratio (HR) supposing continuous EPclin score was 1.87 [95% confidence interval (CI) 1.4-2.5, P < 0.0001]. This prognostic effect remained significant when assessed in a Cox model adjusting on tumor size, number of positive nodes and tumor grade (HR 1.52, 95% CI 1.09-2.13, P = 0.0141). The 60-month DMFS for patients in the EPCL and EPCH groups was 100% and 94%, respectively (adjusted HR 8.10, 95% CI 1.1-59.1, P < 0.0001). CONCLUSIONS: The results confirm the value of EPclin score as an independent prognostic parameter in node-positive, hormone receptor-positive, HER2-negative early breast cancer patients receiving standard adjuvant treatment. EPclin score can be used to identify patients at higher risk of recurrence who may warrant additional systemic treatments.

Sphenoid sinus fungall ball: a retrospective study over a 10- year period.

A fungal ball consists of a dense conglomerate of fungal hyphae growing at the surface of the sinus mucosa without tissue infiltration. The maxillary sinus is by far the most commonly involved paranasal sinus cavity followed by the sphenoid sinus. The present study is a retrospective study of 25 consecutive cases treated during the last 10 years in the two hospitals be- longing to the Catholic University of Louvain (CHU Mont-Godinne and UCL Saint Luc). We report the symptomatology, the imaging and discuss the different surgical managements. We conclude that the clinician must have a high index of suspicion when dealing with a unilateral rhinosinusitis persisting despite a maximal and well conducted medical treatment. This is particularly so in elderly women when associated with facial pain and post nasal drip, particularly when the computed tomography shows an unilateral opacity of the sphenoid sinus with or without a sclerosis or an erosion of the bony walls, a polyp in the sphenoethmoidal recess or a hyperdensity mimicking a foreign body. An endonasal endoscopic sphenoidotomy is the treatment of choice in most cases, allowing good ventilation of the sinus and radical removal of all the fungal concretion. A biopsy of the sinus mucosa adjacent to fungal elements is of upmost important to confirm the non- invasiveness of the fungi within the tissue. Antifungal medication is not required in uncomplicated forms. All host factors producing some degree of immunosuppression must be corrected when present and must alert the clinician to rule out any forms of invasive disease.

Alpelisib and fulvestrant in PIK3CA-mutated hormone receptor-positive HER2-negative advanced breast cancer included in the French early access program.

SOLAR-1 and BYLieve trials documented the efficacy of the PI3K-inhibitor alpelisib in pre-treated PIK3CA-mutant, hormone receptor-positive, HER2-negative (HR+/HER2-) advanced breast cancer (ABC) patients. We report here real-life data of patients prospectively registered in the French alpelisib early access program (EAP) opened to PIK3CA-mutant HR+/HER2- ABC patients treated with alpelisib and fulvestrant. Primary endpoint was PFS by local investigators using RECIST1.1. Eleven centers provided individual data on 233 consecutive patients. Patients had received a median number of 4 (range: 1-16) prior systemic treatments for ABC, including CDK4/6 inhibitor, chemotherapy, fulvestrant and everolimus in 227 (97.4%), 180 (77.3%), 175 (75.1%) and 131 (56.2%) patients, respectively. After a median follow-up of 7.1 months and 168 events, median PFS was 5.3 months (95% CI: 4.7-6.0). Among 186 evaluable patients, CBR at 6 months was 45.3% (95% CI: 37.8-52.8). In multivariable analysis, characteristics significantly associated with a shorter PFS were age < 60 years (HR = 1.5, 95% CI = 1.1-2.1), >5 lines of prior treatments (HR = 1.4, 95% CI = 1.0-2.0) and the C420R PI3KCA mutation (HR = 4.1, 95% CI = 1.3-13.6). N = 91 (39.1%) patients discontinued alpelisib due to adverse events. To our knowledge, this is the largest real-life assessment of alpelisib efficacy. Despite heavy pre-treatments, patients derived a clinically relevant benefit from alpelisib and fulvestrant.

Reducing methylmercury accumulation in the food webs of San Francisco Bay and its local watersheds.

San Francisco Bay (California, USA) and its local watersheds present an interesting case study in estuarine mercury (Hg) contamination. This review focuses on the most promising avenues for attempting to reduce methylmercury (MeHg) contamination in Bay Area aquatic food webs and identifying the scientific information that is most urgently needed to support these efforts. Concern for human exposure to MeHg in the region has led to advisories for consumption of sport fish. Striped bass from the Bay have the highest average Hg concentration measured for this species in USA estuaries, and this degree of contamination has been constant for the past 40 years. Similarly, largemouth bass in some Bay Area reservoirs have some of the highest Hg concentrations observed in the entire US. Bay Area wildlife, particularly birds, face potential impacts to reproduction based on Hg concentrations in the tissues of several Bay species. Source control of Hg is one of the primary possible approaches for reducing MeHg accumulation in Bay Area aquatic food webs. Recent findings (particularly Hg isotope measurements) indicate that the decades-long residence time of particle-associated Hg in the Bay is sufficient to allow significant conversion of even the insoluble forms of Hg into MeHg. Past inputs have been thoroughly mixed throughout this shallow and dynamic estuary. The large pool of Hg already present in the ecosystem dominates the fraction converted to MeHg and accumulating in the food web. Consequently, decreasing external Hg inputs can be expected to reduce MeHg in the food web, but it will likely take many decades to centuries before those reductions are achieved. Extensive efforts to reduce loads from the largest Hg mining source (the historic New Almaden mining district) are underway. Hg is spread widely across the urban landscape, but there are a number of key sources, source areas, and pathways that provide opportunities to capture larger quantities of Hg and reduce loads from urban runoff. Atmospheric deposition is a lower priority for source control in the Bay Area due to a combination of a lack of major local sources. Internal net production of MeHg is the dominant source of MeHg that enters the food web. Controlling internal net production is the second primary management approach, and has the potential to reduce food web MeHg in some habitats more effectively and within a much shorter time-frame. Controlling net MeHg production and accumulation in the food web of upstream reservoirs and ponds is very promising due to the many features of these ecosystems that can be manipulated. The most feasible control options in tidal marshes relate to the design of flow patterns and subhabitats in restoration projects. Options for controlling MeHg production in open Bay habitat are limited due primarily to the highly dispersed distribution of Hg throughout the ecosystem. Other changes in these habitats may also have a large influence on food web MeHg, including temperature changes due to global warming, sea level rise, food web alterations due to introduced species and other causes, and changes in sediment supply. Other options for reducing or mitigating exposure and risk include controlling bioaccumulation, cleanup of contaminated sites, and reducing other factors (e.g., habitat availability) that limit at-risk wildlife populations.

Stable isotope mixing models demonstrate the role of an invasive plant in wetland songbird food webs.

Premise: Invasive plants in wetlands are often ecosystem engineers, mediating changes in ecosystem functions like trophic support. We documented the impacts of Lepidium latifolium, an invasive plant, on the food web of omnivorous birds (Suisun song sparrows, Melospiza melodia maxillaris) in a tidal wetland of northern California, USA. Methods: We used analysis of natural abundance stable isotopes of 13C and 15N in song sparrow blood, invertebrate food sources, L. latifolium seeds, and other marsh plant seeds to inform Bayesian, concentration-dependent mixing models that predicted average song sparrow diets. Results: Season and plant phenology influenced food source incorporation and isotopic signatures. Song sparrows showed higher isotopic variability in the summer. The observed changes in song sparrow diets were driven by altered invertebrate communities related to seasonal L. latifolium presence and by shifts from seeds to consumption of invertebrate food sources during the breeding season in the spring and summer. Discussion: This study used stable isotope tools and modeling to demonstrate two mechanisms of isotopic influence by L. latifolium on omnivorous song sparrows. This study can inform site- and species-specific management strategies by demonstrating how changes to the plant community can impact entire trophic systems.

Water quality in South San Francisco Bay, California: current condition and potential issues for the South Bay Salt Pond Restoration Project.

The SBSPRP is an extensive tidal wetland restoration project that is underway at the margin of South San Francisco Bay, California. The Project, which aims to restore former salt ponds to tidal marsh and manage other ponds for water bird support, is taking place in the context of a highly urbanized watershed and an Estuary already impacted by chemical contaminants. There is an intimate relationship between water quality in the watershed, the Bay, and the transitional wetland areas where the Project is located. The Project seeks to restore habitat for endangered and endemic species and to provide recreational opportunities for people. Therefore, water quality and bioaccumulation of contaminants in fish and wildlife is an important concern for the success of the Project. Mercury, PCBs, and PBDEs are the persistent contaminants of greatest concern in the region. All of these contaminants are present at elevated concentrations both in the abiotic environment and in wildlife. Dioxins, pyrethroids, PAHs, and selenium are also problematic. Organochlorine insecticides have historically impacted the Bay, and they remain above thresholds for concern in a small proportion of samples. Emerging contaminants, such as PFCs and non-PBDE flame retardants, are also an important water quality issue. Beyond chemical pollutants, other concerns for water quality in South San Francisco Bay exist, and include biological constituents, especially invasive species, and chemical attributes, such as dissolved oxygen and salinity. Future changes, both from within the Project and from the Bay and watershed, are likely to influence water quality in the region. Project actions to restore wetlands could worsen, improve, or not affect the already impaired water quality in South Bay. Accelerated erosion of buried sediment as a consequence of Project restoration actions is a potentially serious regional threat to South Bay water and sediment quality. Furthermore, the planned restoration of salt ponds to tidal marsh has raised concerns about possible increased net production of methylmercury and its subsequent accumulation in the food web. This concern applies not only to the restored marshes, but also to the South Bay as a whole, which could be affected on a regional scale. The ponds that are converted to tidal marsh will sequester millions of cubic meters of sediment. Sequestration of sediment in marshes could remove contaminated sediment from the active zone of the Bay but could also create marshes with contaminated food webs. Some of the ponds will not be restored to marsh but will be managed for use by water birds. Therefore, the effect of dense avian populations on eutrophication and the introduction of pathogens should be considered. Water quality in the Project also could be affected by external changes, such as human population growth and climate change. To address these many concerns related to water quality, the SBSPRP managers, and others faced with management of wetland restoration at a regional scale, should practice adaptive management and ongoing monitoring for water quality, particularly monitoring bioaccumulation of contaminants in the food web.

Microfilamentous system and secretion of enzyme in the exocrine pancreas. Effect of cytochalasin B.

The microfilaments in the acinar cell of the exocrine pancreas are essentially located in the apical part of the cell: thin microfilaments (50 A), cytochalasin B (CB)-sensitive, form the axis of the microvilli and a network lying beneath the apical membrane; thicker filaments (100 A), at least partly CB-insensitive, form bundles parallel to the plasma cell membrane and the desmosomal links. CB interaction with the acinar cell of the exocrine pancreas involves at least two sites: a membrane site involved in the inhibitory effect of CB on the monosaccharide transport and a less sensitive site at the filamentous level at least partly responsible for the inhibitory effect of CB in the secretion of the exportable enzyme from the pancreatic cell. CB did not alter the energy balance of the acinar cell nor the exchanges of 15-Ca between the extracellular medium and the pancreatic tissue. CB (2 times 10-minus 7 and 2 times 10-minus 6 M) has secretagogue properties whereas CB (2 times 10-minus 5 M) has inhibitory effect on stimulated secretion and secretagogue properties. The mechanism of these secretory effects is not yet explained. The analysis presented in this investigation affords strong evidence for the involvement of the microfilamentous network in the last steps of the secretory cycle in the acinar cell of the exocrine pancreas.

Electrochemical fluorination of La(2)CuO(4): a mild "chimie douce" route to superconducting oxyfluoride materials.

The fluorination of La(2)CuO(4) was achieved for the first time under normal conditions of pressure and temperature (1 MPa and 298 K) via electrochemical insertion in organic fluorinated electrolytes and led to lanthanum oxyfluorides of general formula La(2)CuO(4)F(x). Analyses showed that, underneath a very thin layer of LaF(3) (a few atomic layers), fluorine is effectively inserted in the material's structure. The fluorination strongly modifies the lanthanum environment, whereas very little modification is observed on copper, suggesting an insertion in the La(2)O(2) blocks of the structure. In all cases, fluorine insertion breaks the translation symmetry and introduces a long-distance disorder, as shown by electron spin resonance. These results highlight the efficiency of electrochemistry as a new "chimie douce" type fluorination technique for solid-state materials. Performed at room temperature, it additionally does not require any specific experimental care. The choice of the electrolytic medium is crucial with regard to the fluorine insertion rate as well as the material deterioration. Successful application of this technique to the well-known La(2)CuO(4) material provides a basis for further syntheses from other oxides.

Ki67 Labelling Index of Neoplastic Epithelial Cells Differentiates Canine Acanthomatous Ameloblastoma from Oral Squamous Cell Carcinoma.

Canine acanthomatous ameloblastoma (CAA) and oral squamous cell carcinoma (OSCC) are the most common oral tumours of epithelial origin in dogs. Overlapping clinical, radiographical and histological features can make distinction between CAA and OSCC difficult. The ability to distinguish tumour identity is critical due to their different biological behaviour and recommended treatment modalities, as well as respective comparative and translational applications as potential models of human disease. Based on marked differences in biological behaviour (i.e. benign versus malignant), it is reasonable to predict that the tumour cell proliferation activity is lower in CAA than in OSCC. However, to our knowledge, the epithelial cell proliferation activity of CAA has not been studied or compared with that of OSCC. Therefore, the aims of this study were to (1) compare the neoplastic epithelial cell proliferation activity of CAA and OSCC based on conventional mitotic index (MI) and Ki67 labelling index (LI), and (2) correlate these findings with clinical parameters including patient signalment, anatomical tumour location and degree of local invasion at the time of diagnosis as determined by computed tomography. We found that (1) the Ki67 LI of OSSC (n = 14) was significantly higher than that of CAA (n = 25), (2) the Ki67 LI correlated with a more aggressive locally invasive behaviour, and (3) the MI was not associated with tumour type. We conclude that the Ki67 LI, but not the MI, is a useful differential marker of CAA from OSCC, and that the epithelial cell proliferation activities of OSCC and CAA correlate with their known differences in biological behaviour.

Evolution of the entire arthropod Hox gene set predated the origin and radiation of the onychophoran/arthropod clade.

BACKGROUND: Dramatic changes in body size and pattern occurred during the radiation of many taxa in the Cambrian, and these changes are best documented for the arthropods. The sudden appearance of such diverse body plans raises the fundamental question of when the genes and the developmental control systems that regulate these designs evolved. As Hox genes regulate arthropod body patterns, the evolution of these genes may have played a role in the origin and diversification of the arthropod body plan from a homonomous ancestor. To trace the origin of arthropod Hox genes, we examined their distribution in a myriapod and in the Onychophora, a sister group to the arthropods. RESULTS: Despite the limited segmental diversity within myriapods and Onychophora, all insect Hox genes are present in both taxa, including the trunk Hox genes Ultrabithorax and abdominal-A as well as an ortholog of the fushi tarazu gene. Comparative analysis of Hox gene deployment revealed that the anterior boundary of expression of trunk Hox genes has shifted dramatically along the anteroposterior axis between Onychophora and different arthropod classes. Furthermore, we found that repression of expression of the Hox target gene Distal-less is unique to the insect lineage. CONCLUSIONS: A complete arthropod Hox gene family existed in the ancestor of the onychophoran/arthropod clade. No new Hox genes were therefore required to catalyze the arthropod radiation; instead, arthropod body-plan diversity arose through changes in the regulation of Hox genes and their downstream targets.

Chick embryo pancreatic transplants reverse experimental diabetes of rats.

The effectiveness of xenogeneic embryonic tissue in the treatment of experimental diabetes has been investigated in rats. The splenic lobes (80) of 15- to 18-d-old chick embryos, composed almost exclusively of endocrine tissue, were implanted directly into the hepatic parenchyma of the rat recipient. The biochemical and metabolic changes in the recipients suggest that embryonic transplants of 15-d-old chick pancreases were able to significantly improve, for a prolonged period of time (18 mo), the diabetic state of nonimmunosuppressed rats. None of the recipients of 18-d-old embryos splenic lobes exhibited a long-term improvement of the diabetic state after transplantation. The complete destruction of the pancreatic B cells of the recipients was assessed by: (a) immunocytochemical investigations of the recipient's pancreas, (b) measurement of insulin in the liver and pancreas of the recipients and (c) in situ vascular perfusion of their pancreas submitted to high glucose challenge. The results suggest that pancreatic tissue of the 15-d-old embryos is immunologically immature lacking one or several lymphocyte subsets implicated in the afferent lood of "non-self" recognition.

Reliability and discriminant validity of HER2 gene quantification and chromosome 17 aneusomy analysis by real-time PCR in primary breast cancer.

There is an increasing demand for the evaluation of HER2 status in breast cancer. In this study, sections from fixed tissues and triton extracts of tissue homogenates were obtained from 163 malignant breast tumors and analyzed in parallel using immunohistochemistry combined with fluorescence in situ hybridization, as gold standard tests, and an ELISA test (c-erbB2/c-neu Rapid Format ELISA, Oncogene Research Products, USA). Tumor DNA was employed to evaluate two quantitative PCR methods: the HER2/neu DNA Quantification Kit (Roche Diagnostics GmbH, Germany), which uses the gastrin chromosome 17 reference gene, and our recently developed Oncolab qPCR assay, where both a chromosome 17 gene (somatostatin receptor type II (SSTR2)) and a non-chromosome 17 reference gene (glyceraldehyde-3-phosphate deshydrogenase (GAPDH)) were used to detect an increase in HER2 gene copy number and to evaluate the aneusomy of chromosome 17, respectively. By IHC/FISH and ELISA, HER2 was overexpressed in 27 (16.6%) and 24 (14.7%) samples, respectively. With the Roche and Oncolab qPCR assays, 29 (17.8%) samples showed a ratio of HER2/gastrin > or = 2.0 and 26 (16.0%) showed a ratio of HER2/SSTR2 > or = 2.0, respectively. In samples presenting HER2/SSTR2 <2.0 and HER2/GAPDH > or = 2.0, which was indicative of a chromosome 17 polysomy, we observed a modest increase in HER2 protein expression. Complete agreement between the four methods for HER2 status determination was obtained for 154 (94.5%) samples. Overall, these results demonstrate that quantitative PCR is a reliable method for analyzing HER2 status and chromosome 17 polysomy.

A homogeneous europium cryptate-based assay for the diagnosis of mutations by time-resolved fluorescence resonance energy transfer.

Oligonucleotide ligation assay (OLA) is considered to be a very useful methodology for the detection and characterization of mutations, particularly for clinical purposes. The fluorescence resonance energy transfer between a fluorescent donor and a suitable fluorophore as acceptor has been applied in the past to several scientific fields. This technique is well adapted to nucleic acid analysis such as DNA sequencing, DNA hybridization and polymerase chain reaction. We describe here a homogeneous format based on the use of a rare earth cryptate label as donor: tris-bipyridine-Eu(3+). The long-lived fluorescence of this label makes it possible to reach a high sensitivity by using a time-resolved detection mode. A non-radiative energy transfer technology, known as time-resolved amplification of cryptate emission (TRACE((R))) characterized by a temporal and spectral selectivity has been developed. The TRACE((R)) detection of characterized single nucleotide polymorphism using the OLA for allelic discrimination is proposed. We demonstrate the potentialities of this OLA-TRACE((R)) methodology through the analysis of K-ras oncogene point mutations.

Serum fragment of cytokeratin subunit 19 measured by CYFRA 21-1 immunoradiometric assay as a marker of lung cancer.

Cytokeratin 19 is a subunit of cytokeratin intermediate filament expressed in simple epithelia and their malignant counterparts. Therefore, it is expressed by respiratory epithelium cells and has been detected in lung cancer specimens. An immunoradiometric assay was used to detect a fragment of the cytokeratin 19, referred to as CYFRA 21-1, in the serum of 165 patients with histologically proved lung cancer (128 non-small cell and 37 small cell lung cancers). This prospective study was conducted to evaluate the reliability of this immunoradiometric assay and to identify the relationship between serum CYFRA 21-1 and different features of lung cancer including prognosis. The minimal detectable concentration detected by this assay was 0.06 ng/ml. The reliability of the immunoradiometric assay was demonstrated by the linear relationship between CYFRA 21-1 measurement and dilution of the serum, the reproducibility of the dosage in intraassay and interassay, and the high sensitivity of the method in discriminating low CYFRA 21-1 concentrations. Using a threshold of 3.6 ng/ml, sensitivity and specificity were 0.52 and 0.87, respectively. The sensitivity of the marker was highest in squamous cell carcinoma and lowest in small cell carcinoma. In non-small cell lung cancer patients, the marker varied significantly according to both stage of the disease (Kruskal-Wallis, 13.7; P < 0.005) and performance status (Kruskal-Wallis, 9.16; P < 0.05) inasmuch as a high serum CYFRA 21-1 level was associated with advanced stages, mediastinal lymph nodes, and poor performance status. Consequently, the marker was significantly lower in patients who were operated upon when compared with unresectable ones. Lung cancer patients with serum CYFRA 21-1 over 3.6 ng/ml proved to have a significantly shorter overall survival than those with a normal serum level (log rank, P = 0.007; Wilcoxon, P = 0.001). The negative prognostic effect of CYFRA 21-1 was highly significant in squamous cell carcinomas whereas it was nonsignificant for the other histologies. In Cox's model analysis, performance status, stage grouping, and CYFRA 21-1 were the only significant determinants of survival. This study supports the use of the serum fragment of cytokeratin subunit 19 CYFRA 21-1 as an independent prognostic marker of squamous cell carcinoma of the lung.