Development, Validation, and Clinical Utility of a Six-gene Signature to Predict Aggressive Prostate Cancer.

BACKGROUND: Molecular signatures in prostate cancer (PCa) tissue can provide useful prognostic information to improve the understanding of a patient's risk of harbouring aggressive disease. OBJECTIVE: To develop and validate a gene signature that adds independent prognostic information to clinical parameters for better treatment decisions and patient management. DESIGN, SETTING, AND PARTICIPANTS: Expression of 14 genes was evaluated in radical prostatectomy (RP) tissue from an Irish cohort of PCa patients (n = 426). A six-gene molecular risk score (MRS) was identified with strong prognostic performance to predict adverse pathology (AP) at RP or biochemical recurrence (BCR). The MRS was combined with the Cancer of the Prostate Risk Assessment (CAPRA) score, to create a molecular and clinical risk score (MCRS), and validated in a Swedish cohort (n = 203). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The primary AP outcome was assessed by the likelihood ratio statistics and area under the receiver operating characteristics curves (AUC) from logistic regression models. The secondary time to BCR outcome was assessed by likelihood ratio statistics and C-indexes from Cox proportional hazard regression models. RESULTS AND LIMITATIONS: The six-gene signature was significantly (p < 0.0001) prognostic and added significant prognostic value to clinicopathological features for AP and BCR outcomes. For both outcomes, both the MRS and the MCRS increased the AUC/C-index when added to European Association of Urology (EAU) and CAPRA scores. Limitations include the retrospective nature of this study. CONCLUSIONS: The six-gene signature has strong performance for the prediction of AP and BCR in an independent clinical validation study. MCRS improves prognostic evaluation and can optimise patient management after RP. PATIENT SUMMARY: We found that the expression panel of six genes can help predict whether a patient is likely to have a disease recurrence after radical prostatectomy surgery.

Apoptosis and autophagy markers predict survival in neoadjuvant treated oesophageal adenocarcinoma patients.

BACKGROUND: Less than 20 % of patients with resectable oesophageal adenocarcinoma obtain a pathological response following neoadjuvant chemotherapy. Studies using oesophageal cancer cell lines have shown that drug sensitive tumour cells undergo apoptosis in response to drug treatment, whereas resistant cells induce autophagy and can recover following withdrawal of drug. In this study, we evaluated markers of apoptosis (active/cleaved caspase-3) and autophagy (LC3B) to establish whether these markers are useful prognostic indicators following neoadjuvant therapy. METHODS: Oesophageal adenocarcinoma tumour tissue from the Northern Ireland Biobank at Queens University Belfast was examined retrospectively. Tumours from 144 patients treated with platinum-based neoadjuvant chemotherapy followed by surgical resection were assembled into tissue microarrays prior to immunohistochemical analysis. Kaplan-Meier survival curves and log-rank tests were used to assess the impact of cleaved caspase-3 and LC3B expression on survival. Cox regression was used to examine association with clinical risk factors. RESULTS: High levels of cleaved caspase-3 were found in 14.6 % of patients and this correlated with a significantly better overall survival (p = 0.03). 38.9 % of patients had high cytoplasmic LC3B expression, which correlated with poor overall survival (p = 0.041). In addition, a distinct globular pattern of LC3B expression was identified in 40.3 % of patients and was also predictive of overall survival (p < 0.001). LC3B globular structures are also associated with tumour recurrence (p = 0.014). When these markers were assessed in combination, it was found that patients who showed low/negative cleaved caspase-3 staining and high/positive staining for both patterns of LC3B had the worst overall survival (p < 0.001). Multi-variate analysis also indicated that this marker combination was an independent predictor of poor prognosis (p = 0.008; HR = 0.046, 95% CI = (0.005-0.443). CONCLUSIONS: The expression of cleaved caspase-3 and specific LC3B staining patterns are associated with overall survival following neoadjuvant treatment. The combination of these markers is an independent indicator of outcome in neoadjuvant chemotherapy treated oesophageal adenocarcinoma.

Genetic Variation in Flowering Traits of Tasmanian Leptospermum scoparium and Association with Provenance Home Site Climatic Factors.

Leptospermum scoparium is emerging as an economically important plant for the commercial production of manuka honey and essential oils, both exhibiting unique antibacterial attributes. To support its domestication this is the first quantitative genetic study of variation for L. scoparium traits. It utilised plants from 200 open-pollinated families derived from 40 native populations, from across the species range in Tasmania, grown in a common garden field trial. The traits studied were survival, growth, and the flowering traits precocity, the timing of seasonal peak flowering, flowering duration, and flowering intensity. Significant genetic variation was evident at the population level for all traits studied and at the family level for three traits-growth, flowering precocity, and time to peak flowering. These three traits had moderate to high narrow-sense heritability estimates ranging from 0.27 to 0.69. For six of the traits studied, population differences were associated with climate attributes at the locations where seed was collected, suggesting adaptation to the local climate may have contributed to the observed population differentiation. Population level geographical trends suggest that genotypes to focus on for domestication originate from the eastern half of Tasmania for precociousness and the western half of Tasmania for earlier time to peak flowering and extended flowering duration.

Analytical Validation of a Novel 6-Gene Signature for Prediction of Distant Recurrence in Estrogen Receptor-Positive, HER2-Negative, Early-Stage Breast Cancer.

BACKGROUND: OncoMasTR is a recently developed multigene prognostic test for early-stage breast cancer. The test has been developed in a kit-based format for decentralized deployment in molecular pathology laboratories. The analytical performance characteristics of the OncoMasTR test are described in this study. METHODS: Expression levels of 6 genes were measured by 1-step reverse transcription-quantitative PCR on RNA samples prepared from formalin-fixed, paraffin-embedded (FFPE) breast tumor specimens. Assay precision, reproducibility, input range, and interference were determined using FFPE-derived RNA samples representative of low and high prognostic risk scores. A pooled RNA sample derived from 6 FFPE breast tumor specimens was used to establish the linear range, limit of detection, and amplification efficiency of the individual gene expression assays. RESULTS: The overall precision of the OncoMasTR test was high with an SD of 0.16, which represents less than 2% of the 10-unit risk score range. Test results were reproducible across 4 testing sites, with correlation coefficients of 0.94 to 0.96 for the continuous risk score and concordance of 86% to 96% in low-/high-risk sample classification. Consistent risk scores were obtained across a > 100-fold RNA input range. Individual gene expression assays were linear up to quantification cycle values of 36.0 to 36.9, with amplification efficiencies of 80% to 102%. Test results were not influenced by agents used during RNA isolation, by low levels of copurified genomic DNA, or by moderate levels of copurified adjacent nontumor tissue. CONCLUSION: The OncoMasTR prognostic test displays robust analytical performance that is suitable for deployment by local pathology laboratories for decentralized use.

Prognostic value of the 6-gene OncoMasTR test in hormone receptor-positive HER2-negative early-stage breast cancer: Comparative analysis with standard clinicopathological factors.

AIM: The aim of the study was to assess the prognostic performance of a 6-gene molecular score (OncoMasTR Molecular Score [OMm]) and a composite risk score (OncoMasTR Risk Score [OM]) and to conduct a within-patient comparison against four routinely used molecular and clinicopathological risk assessment tools: Oncotype DX Recurrence Score, Ki67, Nottingham Prognostic Index and Clinical Risk Category, based on the modified Adjuvant! Online definition and three risk factors: patient age, tumour size and grade. METHODS: Biospecimens and clinicopathological information for 404 Irish women also previously enrolled in the Trial Assigning Individualized Options for Treatment [Rx] were provided by 11 participating hospitals, as the primary objective of an independent translational study. Gene expression measured via RT-qPCR was used to calculate OMm and OM. The prognostic value for distant recurrence-free survival (DRFS) and invasive disease-free survival (IDFS) was assessed using Cox proportional hazards models and Kaplan-Meier analysis. All statistical tests were two-sided ones. RESULTS: OMm and OM (both with likelihood ratio statistic [LRS] P < 0.001; C indexes = 0.84 and 0.85, respectively) were more prognostic for DRFS and provided significant additional prognostic information to all other assessment tools/factors assessed (all LRS P ≤ 0.002). In addition, the OM correctly classified more patients with distant recurrences (DRs) into the high-risk category than other risk classification tools. Similar results were observed for IDFS. DISCUSSION: Both OncoMasTR scores were significantly prognostic for DRFS and IDFS and provided additional prognostic information to the molecular and clinicopathological risk factors/tools assessed. OM was also the most accurate risk classification tool for identifying DR. A concise 6-gene signature with superior risk stratification was shown to increase prognosis reliability, which may help clinicians optimise treatment decisions.

Development of a Novel Weighted Ranking Method for Immunohistochemical Quantification of a Heterogeneously Expressed Protein in Gastro-Esophageal Cancers.

High expression of Junctional Adhesion Molecule-A (JAM-A) has been linked with poor prognosis in several cancers, including breast cancers overexpressing the human epidermal growth factor receptor-2 (HER2). Furthermore, JAM-A expression has been linked with regulating that of HER2, and associated with the development of resistance to HER2-targeted therapies in breast cancer patients. The purpose of this study was to establish a potential relationship between JAM-A and HER2 in HER2-overexpressing gastro-esophageal (GE) cancers. Interrogation of gene expression datasets revealed that high JAM-A mRNA expression was associated with poorer survival in HER2-positive gastric cancer patients. However, high intra-tumoral heterogeneity of JAM-A protein expression was noted upon immunohistochemical scoring of a GE cancer tissue microarray (TMA), precluding a simple confirmation of any relationship between JAM-A and HER2 at protein level. However, in a test-set of 25 full-face GE cancer tissue sections, a novel weighted ranking system proved effective in capturing JAM-A intra-tumoral heterogeneity and confirming statistically significant correlations between JAM-A/HER2 expression. Given the growing importance of immunohistochemistry in stratifying cancer patients for the receipt of new targeted therapies, this may sound a cautionary note against over-relying on cancer TMAs in biomarker discovery studies of heterogeneously expressed proteins. It also highlights a timely need to develop validated mechanisms of capturing intra-tumoral heterogeneity to aid in future biomarker/therapeutic target development for the benefit of cancer patients.

Identification and clinical impact of potentially actionable somatic oncogenic mutations in solid tumor samples.

BACKGROUND: An increasing number of anti-cancer therapeutic agents target specific mutant proteins that are expressed by many different tumor types. Successful use of these therapies is dependent on the presence or absence of somatic mutations within the patient's tumor that can confer clinical efficacy or drug resistance. METHODS: The aim of our study was to determine the type, frequency, overlap and functional proteomic effects of potentially targetable recurrent somatic hotspot mutations in 47 cancer-related genes in multiple disease sites that could be potential therapeutic targets using currently available agents or agents in clinical development. RESULTS: Using MassArray technology, of the 1300 patient tumors analysed 571 (43.9%) had at least one somatic mutation. Mutations were identified in 30 different genes. KRAS (16.5%), PIK3CA (13.6%) and BRAF (3.8%) were the most frequently mutated genes. Prostate (10.8%) had the lowest number of somatic mutations identified, while no mutations were identified in sarcoma. Ocular melanoma (90.6%), endometrial (72.4%) and colorectal (66.4%) tumors had the highest number of mutations. We noted high concordance between mutations in different parts of the tumor (94%) and matched primary and metastatic samples (90%). KRAS and BRAF mutations were mutually exclusive. Mutation co-occurrence involved mainly PIK3CA and PTPN11, and PTPN11 and APC. Reverse Phase Protein Array (RPPA) analysis demonstrated that PI3K and MAPK signalling pathways were more altered in tumors with mutations compared to wild type tumors. CONCLUSIONS: Hotspot mutational profiling is a sensitive, high-throughput approach for identifying mutations of clinical relevance to molecular based therapeutics for treatment of cancer, and could potentially be of use in identifying novel opportunities for genotype-driven clinical trials.

External quality assessment demonstrates that PD-L1 22C3 and SP263 assays are systematically different.

PD-L1 inhibitors are part of first line treatment options for patients with advanced non-small cell lung cancer. PD-L1 immunohistochemistry (IHC) assays act as either a companion or a complementary diagnostic. The purpose of this study is to describe the experience of external quality assurance (EQA) provider UK NEQAS ICC and ISH with the comparison of different PD-L1 assays used in daily practice. Three EQA rounds (pilot, run A and run B) were carried out using formalin fixed paraffin embedded samples with sample sets covering a range of epitope concentrations, including 'critical samples' near to clinical threshold cut-offs. An expert panel (n = 4) evaluated all returned slides simultaneously and independently on a multi-header microscope together with the participants own in-house control material. The tonsil sample was evaluated as 'acceptable' or 'unacceptable', and for the other samples the percentage of PD-L1 stained tumour cells were estimated in predetermined categories (<1%, 1 to <5%, 5 to <10%, 10 to <25%, 25 to <50%, 50 to <80%, 80 to 100%). In the pilot and the two subsequent runs the number of participating laboratories was 43, 69 and 76, respectively. The pass rate for the pilot run was 67%; this increased to 81% at run A and 82% at run B. For two 'critical samples', in runs A and B, 22C3 IHC had significantly higher PD-L1 expression than SP263 IHC (p < 0.001), whilst the PD-L1 scores for the other six samples were similar for all assays. In run A the laboratory developed tests (LDTs) using 22C3 scored lower than the commercial 22C3 tests (p = 0.01). After the initial testing, improvement in performance of PD-L1 IHC is shown for approved and LDT PD-L1 assays. Equivalency of approved PD-L1 22C3 and SP263 assays cannot be assumed as the scores cross the clinically relevant thresholds of 1% and 50% PD-L1 expression.

Trees on farms to support natural capital: An evidence-based review for grazed dairy systems.

Intensification of the dairy industry globally, combined with a changing climate, has placed increased pressure on natural capital assets (and the flow of ecosystem services) on farms. Agroforestry is widely promoted as an intervention to address these issues. While some benefits of integrating trees on farms, such as carbon sequestration and biodiversity, are reasonably well known, less is known about other potential benefits, such as on-farm production. Understanding and quantifying these benefits would inform farm planning and decision-making. We used a systematic review approach to analyse the evidence base for biophysical ecosystem services from woody systems (including shelterbelts, riparian plantings, plantations, pasture trees, silvopasture and remnant native vegetation) provided to grazed dairy enterprises. We identified 83 publications containing 123 records that fit our review criteria of reporting on biophysical ecosystem services from woody systems on dairy farms relative to a grazed pasture comparison. For each relationship between a woody system and ecosystem service, we assessed the level of support, strength and predominant direction of evidence, and summarised the causal relationships (woody system ≫ mechanism ≫ outcome). Shelterbelts and riparian plantings were the most commonly reported woody systems. Linkages between woody systems and ecosystem services were largely positive, with the types of services provided and their importance differing among systems. Mean evaluation scores for the strength of the evidence were moderate to strong. However, the number of records for each relationship was often low. Consequently, only eight of the 30 causal pathways identified had high confidence; a further 14 had medium confidence indicating that these have good potential to deliver benefits but warrant further work. Although the evidence here was largely qualitative, our results provide strong support for the internal benefits that natural capital assets, such as on-farm woody systems, can provide to the productivity and resilience of grazed dairy enterprises.

Correction to: LC3B globular structures correlate with survival in esophageal adenocarcinoma.

Following publication of the original article [1], the authors reported an omission in the affiliations.

Drought response strategies and hydraulic traits contribute to mechanistic understanding of plant dry-down to hydraulic failure.

Drought-induced tree mortality alters forest structure and function, yet our ability to predict when and how different species die during drought remains limited. Here, we explore how stomatal control and drought tolerance traits influence the duration of drought stress leading to critical levels of hydraulic failure. We examined the growth and physiological responses of four woody plant species (three angiosperms and one conifer) representing a range of water-use and drought tolerance traits over the course of two controlled drought-recovery cycles followed by an extended dry-down. At the end of the final dry-down phase, we measured changes in biomass ratios and leaf carbohydrates. During the first and second drought phases, plants of all species closed their stomata in response to decreasing water potential, but only the conifer species avoided water potentials associated with xylem embolism as a result of early stomatal closure relative to thresholds of hydraulic dysfunction. The time it took plants to reach critical levels of water stress during the final dry-down was similar among the angiosperms (ranging from 39 to 57 days to stemP88) and longer in the conifer (156 days to stemP50). Plant dry-down time was influenced by a number of factors including species stomatal-hydraulic safety margin (gsP90 - stemP50), as well as leaf succulence and minimum stomatal conductance. Leaf carbohydrate reserves (starch) were not depleted at the end of the final dry-down in any species, irrespective of the duration of drought. These findings highlight the need to consider multiple structural and functional traits when predicting the timing of hydraulic failure in plants.

Estrogen receptor-alpha isoforms are the main estrogen receptors expressed in non-small cell lung carcinoma.

The expression profile of estrogen receptors (ER) in Non-Small Cell Lung Carcinoma (NSCLC) remains contradictory. Here we investigated protein and transcriptome expression of ERα wild type and variants. Tissue Micro-Arrays of 200 cases of NSCLC (paired tumor/non-tumor) were assayed by immunohistochemistry using a panel of ERα antibodies targeting different epitopes (HC20, 6F11, 1D5, ERα36 and ERα17p). ERß epitopes were also examined for comparison. In parallel we conducted a probe-set mapping (Affymetrix HGU133 plus 2 chip) meta-analysis of 12 NSCLC tumor public transcriptomic studies (1418 cases) and 39 NSCLC cell lines. Finally, we have investigated early transcriptional effects of 17ß-estradiol, 17ß-estradiol-BSA, tamoxifen and their combination in two NSCLC cell lines (A549, H520). ERα transcript and protein detection in NSCLC specimens and cell lines suggests that extranuclear ERα variants, like ERα36, prevail, while wild-type ERα66 is minimally expressed. In non-tumor lung, the wild-type ERα66 is quasi-absent. The combined evaluation of ERα isoform staining intensity and subcellular localization with sex, can discriminate NSCLC subtypes and normal lung. Overall ERα transcription decreases in NSCLC. ERα expression is sex-related in non-tumor tissue, but in NSCLC it is exclusively correlating with tumor histologic subtype. ERα isoform protein expression is higher than ERß. ERα isoforms are functional and display specific early transcriptional effects following steroid treatment. In conclusion, our data show a wide extranuclear ERα-variant expression in normal lung and NSCLC that is not reported by routine pathology ER evaluation criteria, limited in the nuclear wild type receptor.

Responses of riparian forests to flood irrigation in the hyper-arid zone of NW China.

Knowledge of forest water use is crucial to water resources managers, especially in arid environments. Flood irrigation has sometimes been used to ameliorate forest decline, however, there has only been limited research on vegetation responses to these interventions. We undertook a study to quantify evapotranspiration (ET) and its components, transpiration (T) and evaporation (E), of two Populus euphratica Oliv. stands (MA: middle-aged and OA: old-aged) with and without flood irrigation in the lower Heihe River Basin of NW China. ET and T were measured using eddy covariance and sap flow methods, respectively. Understory E was estimated by difference. Annual ET was 766.4 mm in the MA stand and 532.5 mm in the OA stand with an average of 4.2 and 2.9 mm d-1 during the growing season, respectively. ET of the MA stand was 44% higher than that of the OA stand, with contributions of 28% and 16% from E and T. Despite stand density, leaf area index and canopy cover being higher in the MA than OA stand sapwood area within the two stands was similar (MA 6.04 m2 ha-1 and OA 6.02 m2 ha-1). We hypothesised lower understory E and a lower E to ET ratio in the MA stand than OA stand. However, E was approximately 63% of ET in both stands. Therefore, we conclude that differences in ET, T and E were mainly associated with the flood irrigation. This was further supported by the comparable ET between the OA stand and the other studies in arid regions of Central Asia. In conclusion, flood irrigation has a less significant effect on canopy water use (T) than understory E suggesting alternatives to flood irrigation might be more appropriate in this water-limited ecosystem.

Stromal TRIM28-associated signaling pathway modulation within the colorectal cancer microenvironment.

BACKGROUND: Stromal gene expression patterns predict patient outcomes in colorectal cancer. TRIM28 is a transcriptional co-repressor that regulates an abundance of genes through the KRAB domain family of transcription factors. We have previously shown that stromal expression of TRIM28 is a marker of disease relapse and poor survival in colorectal cancer. Here, we perform differential epithelium-stroma proteomic network analyses to characterize signaling pathways associated with TRIM28 within the tumor microenvironment. METHODS: Reverse phase protein arrays were generated from laser capture micro-dissected carcinoma and stromal cells from fresh frozen colorectal cancer tissues. Phosphorylation and total protein levels were measured for 30 cancer-related signaling pathway endpoints. Strength and direction of associations between signaling endpoints were identified using Spearman's rank-order correlation analysis and compared to TRIM28 levels. Expression status of TRIM28 in tumor epithelium and stromal fibroblasts was assessed using IHC in formalin fixed tissue and the epithelium to stroma protein expression ratio method. RESULTS: We found distinct proteomic networks in the epithelial and stromal compartments which were linked to expression levels of TRIM28. Low levels of TRIM28 in tumor stroma (high epithelium: stroma ratio) were found in 10 out of 19 cases. Upon proteomic network analyses, these stromal high ratio cases revealed moderate signaling pathway similarity exemplified by 76 significant Spearman correlations (ρ ≥ 0.75, p ≤ 0.01). Furthermore, low levels of stromal TRIM28 correlated with elevated MDM2 levels in tumor epithelium (p = 0.01) and COX-2 levels in tumor stroma (p = 0.002). Low TRIM28 epithelium to stroma ratios were associated with elevated levels of caspases 3 and 7 in stroma (p = 0.041 and p = 0.036) and an increased signaling pathway similarity in stromal cells with 81 significant Spearman correlations (ρ ≥ 0.75, p ≤ 0.01). CONCLUSIONS: By dissecting TRIM28-associated pathways in stromal fibroblasts and epithelial tumor cells, we performed comprehensive proteomic analyses of molecular networks within the tumor microenvironment. We found modulation of several signaling pathways associated with TRIM28, which may be attributed to the pleiotropic properties of TRIM28 through its translational suppression of the family of KRAB domain transcription factors in tumor stromal compartments.

Elevated CO2 did not affect the hydrological balance of a mature native Eucalyptus woodland.

Elevated atmospheric CO2 concentration (eCa ) might reduce forest water-use, due to decreased transpiration, following partial stomatal closure, thus enhancing water-use efficiency and productivity at low water availability. If evapotranspiration (Et ) is reduced, it may subsequently increase soil water storage (ΔS) or surface runoff (R) and drainage (Dg ), although these could be offset or even reversed by changes in vegetation structure, mainly increased leaf area index (L). To understand the effect of eCa in a water-limited ecosystem, we tested whether 2 years of eCa (~40% increase) affected the hydrological partitioning in a mature water-limited Eucalyptus woodland exposed to Free-Air CO2 Enrichment (FACE). This timeframe allowed us to evaluate whether physiological effects of eCa reduced stand water-use irrespective of L, which was unaffected by eCa in this timeframe. We hypothesized that eCa would reduce tree-canopy transpiration (Etree ), but excess water from reduced Etree would be lost via increased soil evaporation and understory transpiration (Efloor ) with no increase in ΔS, R or Dg . We computed Et , ΔS, R and Dg from measurements of sapflow velocity, L, soil water content (θ), understory micrometeorology, throughfall and stemflow. We found that eCa did not affect Etree , Efloor , ΔS or θ at any depth (to 4.5 m) over the experimental period. We closed the water balance for dry seasons with no differences in the partitioning to R and Dg between Ca levels. Soil temperature and θ were the main drivers of Efloor while vapour pressure deficit-controlled Etree , though eCa did not significantly affect any of these relationships. Our results suggest that in the short-term, eCa does not significantly affect ecosystem water-use at this site. We conclude that water-savings under eCa mediated by either direct effects on plant transpiration or by indirect effects via changes in L or soil moisture availability are unlikely in water-limited mature eucalypt woodlands.

Prescreening in large populations as a tool for identifying elevated CO2-responsive genotypes in plants.

Elevated atmospheric CO2 concentration (e[CO2]) can stimulate the photosynthesis and productivity of C3 species including food and forest crops. Intraspecific variation in responsiveness to e[CO2] can be exploited to increase productivity under e[CO2]. However, active selection of genotypes to increase productivity under e[CO2] is rarely performed across a wide range of germplasm, because of constraints of space and the cost of CO2 fumigation facilities. If we are to capitalise on recent advances in whole genome sequencing, approaches are required to help overcome these issues of space and cost. Here, we discuss the advantage of applying prescreening as a tool in large genome×e[CO2] experiments, where a surrogate for e[CO2] was used to select cultivars for more detailed analysis under e[CO2] conditions. We discuss why phenotypic prescreening in population-wide screening for e[CO2] responsiveness is necessary, what approaches could be used for prescreening for e[CO2] responsiveness, and how the data can be used to improve genetic selection of high-performing cultivars. We do this within the framework of understanding the strengths and limitations of genotype-phenotype mapping.

A multi-species synthesis of physiological mechanisms in drought-induced tree mortality.

Widespread tree mortality associated with drought has been observed on all forested continents and global change is expected to exacerbate vegetation vulnerability. Forest mortality has implications for future biosphere-atmosphere interactions of carbon, water and energy balance, and is poorly represented in dynamic vegetation models. Reducing uncertainty requires improved mortality projections founded on robust physiological processes. However, the proposed mechanisms of drought-induced mortality, including hydraulic failure and carbon starvation, are unresolved. A growing number of empirical studies have investigated these mechanisms, but data have not been consistently analysed across species and biomes using a standardized physiological framework. Here, we show that xylem hydraulic failure was ubiquitous across multiple tree taxa at drought-induced mortality. All species assessed had 60% or higher loss of xylem hydraulic conductivity, consistent with proposed theoretical and modelled survival thresholds. We found diverse responses in non-structural carbohydrate reserves at mortality, indicating that evidence supporting carbon starvation was not universal. Reduced non-structural carbohydrates were more common for gymnosperms than angiosperms, associated with xylem hydraulic vulnerability, and may have a role in reducing hydraulic function. Our finding that hydraulic failure at drought-induced mortality was persistent across species indicates that substantial improvement in vegetation modelling can be achieved using thresholds in hydraulic function.

Somatic DNA Mutation Analysis.

Somatic mutations in patient tumor DNA samples can be readily detected based on mass spectrometry. The MassARRAY system is a high-throughput matrix-assisted laser desorption time-of-flight (MALDI) mass spectrometer for detection of nucleic acids. The technique is based on single-nucleotide base extension. A series of PCR assays amplify specific DNA regions of interest harboring mutations. A third primer is then introduced into the reaction which corresponds to the DNA template immediately in front of the mutation site. A final round of PCR is then performed using mass-modified nucleotides. These nucleotides are designed so that no additional bases can be added to the extension primer (terminating bases) after a single-base extension and are mass modified to exaggerate mass differences between nucleotides allowing easier identification by mass spectrometry.The sequences of the extension primer and possible extension products (wild type and mutations) are known; therefore, it is possible to calculate their mass. The mass spectrometer can identify the mass peaks for each assay and identify those with mutations (multiple peaks). The technique was originally designed to screen multiple single-nucleotide polymorphisms (SNPs) in a large number of specimens. A SNP in the coding region of DNA that alters the gene and subsequent protein expression is considered a mutation. Mutations often occur in genes whose protein product is in a key signaling pathway and/or drug target. Rationale treatment options can be designed based upon the presence or absence of these mutations. In this chapter, we describe the process for detection of somatic mutations in DNA extracted from formalin-fixed paraffin-embedded (FFPE) material.

An empirical method that separates irreversible stem radial growth from bark water content changes in trees: theory and case studies.

Substantial uncertainty surrounds our knowledge of tree stem growth, with some of the most basic questions, such as when stem radial growth occurs through the daily cycle, still unanswered. We employed high-resolution point dendrometers, sap flow sensors, and developed theory and statistical approaches, to devise a novel method separating irreversible radial growth from elastic tension-driven and elastic osmotically driven changes in bark water content. We tested this method using data from five case study species. Experimental manipulations, namely a field irrigation experiment on Scots pine and a stem girdling experiment on red forest gum trees, were used to validate the theory. Time courses of stem radial growth following irrigation and stem girdling were consistent with a-priori predictions. Patterns of stem radial growth varied across case studies, with growth occurring during the day and/or night, consistent with the available literature. Importantly, our approach provides a valuable alternative to existing methods, as it can be approximated by a simple empirical interpolation routine that derives irreversible radial growth using standard regression techniques. Our novel method provides an improved understanding of the relative source-sink carbon dynamics of tree stems at a sub-daily time scale.

ALK Immunohistochemistry in NSCLC: Discordant Staining Can Impact Patient Treatment Regimen.

INTRODUCTION: Diagnostic immunohistochemistry (IHC) is increasingly accepted as a screening method for anaplastic lymphoma receptor tyrosine kinase gene (ALK) rearrangements in NSCLC. We have sought to establish an ongoing robust external quality assessment process to gauge quality of anaplastic lymphoma kinase (ALK) IHC, which can have an impact on interpretation of patient samples. METHODS: Unstained tissue and cell line samples were distributed on a quarterly basis to participating laboratories from 30 countries. Participants stained the slide using their routine diagnostic ALK IHC method and returned the slide along with their in-house control and methodology details. Slides were assessed by a team of pathologists and scientists. RESULTS: Overall, there was a mean pass rate of 83% (range 71%-98%), with 38 variations in staining protocol. Methods included the following: the Roche D5F3 assay (65% of users, pass rate 93%); Novocastra 5A4 (15% of users, pass rate 65%); Cell Signaling Technology D5F3 (7% of users, pass rate 91%), and Dako ALK1 (5% of users, pass rate 50%). Choice of methodology directly affected final interpretation of distributed ALK-positive and ALK-negative NSCLC cases, which were correctly identified by 89% and 88% of participants, respectively. Antibody detection method was a contributing factor in false-negative staining results. The choice of laboratory controls was found to be unsuitable, and as such, in-house control recommendations are also provided. CONCLUSIONS: ALK IHC is a robust screening technique, but there is concern that some diagnostic laboratories are using inadequate staining methods, which has a direct impact on final interpretation. External assessment helps provide laboratories with continued confidence in their ALK IHC testing.