A Machine Learned Classifier That Uses Gene Expression Data to Accurately Predict Estrogen Receptor Status

Background

Selecting the appropriate treatment for breast cancer requires accurately determining the estrogen receptor (ER) status of the tumor. However, the standard for determining this status, immunohistochemical analysis of formalin-fixed paraffin embedded samples, suffers from numerous technical and reproducibility issues. Assessment of ER-status based on RNA expression can provide more objective, quantitative and reproducible test results.

Methods

To learn a parsimonious RNA-based classifier of hormone receptor status, we applied a machine learning tool to a training dataset of gene expression microarray data obtained from 176 frozen breast tumors, whose ER-status was determined by applying ASCO-CAP guidelines to standardized immunohistochemical testing of formalin fixed tumor.

Results

This produced a three-gene classifier that can predict the ER-status of a novel tumor, with a cross-validation accuracy of 93.17±2.44%. When applied to an independent validation set and to four other public databases, some on different platforms, this classifier obtained over 90% accuracy in each. In addition, we found that this prediction rule separated the patients'' recurrence-free survival curves with a hazard ratio lower than the one based on the IHC analysis of ER-status.

Conclusions

Our efficient and parsimonious classifier lends itself to high throughput, highly accurate and low-cost RNA-based assessments of ER-status, suitable for routine high-throughput clinical use. This analytic method provides a proof-of-principle that may be applicable to developing effective RNA-based tests for other biomarkers and conditions.     

Nicotine up-regulates alpha4beta2 nicotinic receptors and ER exit sites via stoichiometry-dependent chaperoning

The up-regulation of α4β2* nicotinic acetylcholine receptors (nAChRs) by chronic nicotine is a cell-delimited process and may be necessary and sufficient for the initial events of nicotine dependence. Clinical literature documents an inverse relationship between a person’s history of tobacco use and his or her susceptibility to Parkinson’s disease; this may also result from up-regulation. This study visualizes and quantifies the subcellular mechanisms involved in nicotine-induced nAChR up-regulation by using transfected fluorescent protein (FP)-tagged α4 nAChR subunits and an FP-tagged Sec24D endoplasmic reticulum (ER) exit site marker. Total internal reflection fluorescence microscopy shows that nicotine (0.1 µM for 48 h) up-regulates α4β2 nAChRs at the plasma membrane (PM), despite increasing the fraction of α4β2 nAChRs that remain in near-PM ER. Pixel-resolved normalized Förster resonance energy transfer microscopy between α4-FP subunits shows that nicotine stabilizes the (α4)₂(β2)₃ stoichiometry before the nAChRs reach the trans-Golgi apparatus. Nicotine also induces the formation of additional ER exit sites (ERES). To aid in the mechanistic analysis of these phenomena, we generated a β2_{enhanced-ER-export} mutant subunit that mimics two regions of the β4 subunit sequence: the presence of an ER export motif and the absence of an ER retention/retrieval motif. The α4β2_{enhanced-ER-export} nAChR resembles nicotine-exposed nAChRs with regard to stoichiometry, intracellular mobility, ERES enhancement, and PM localization. Nicotine produces only small additional PM up-regulation of α4β2_{enhanced-ER-export} receptors. The experimental data are simulated with a model incorporating two mechanisms: (1) nicotine acts as a stabilizing pharmacological chaperone for nascent α4β2 nAChRs in the ER, eventually increasing PM receptors despite a bottleneck(s) in ER export; and (2) removal of the bottleneck (e.g., by expression of the β2_{enhanced-ER-export} subunit) is sufficient to increase PM nAChR numbers, even without nicotine. The data also suggest that pharmacological chaperoning of nAChRs by nicotine can alter the physiology of ER processes.     

The segmentation clock in mice: interaction between the Wnt and Notch signalling pathways

In the last few years, the efforts to elucidate the mechanisms underlying the segmentation clock in various vertebrate species have multiplied. Early evidence suggested that oscillations are caused by one of the genes under the Notch signalling pathway (like those of the her or Hes families). Recently, Aulehla et al. [Wnt3a plays a major role in the segmentation clock controlling somitogenesis. Dev. Cell 4, 395-406] discovered that Axin2 (a gene under the Wnt3a signalling pathway) also oscillates in the presomitic mesoderm (PSM) of mice embryos and proposed some mechanisms through which the Notch and Wnt3a pathways may interact. They further suggested that a decreasing concentration of Wnt3a along the PSM may be the gradient the segmentation clock interacts with to form somites. These results were reviewed by Rida et al. [A notch feeling of somite segmentation and beyond. Dev. Biol. 265, 2-22], who introduced a complex clockwork comprising genes Hes1, Lfng (under the Notch pathway), and Axin2, as well as their multiple interactions. In the present work we develop a mathematical model based on the Rida et al. review and use it to tackle some of the questions raided by the Aulehla et al. paper: can the Axin2 feedback loop constitute a clock? Could a decreasing Wnt3a signaling constitute the wavefront, where phase is recorded and the spatial pattern laid down? What is the master oscillator?     

Operon dynamics with state dependent transcription and/or translation delays

This paper is focused on SIS (Susceptible-Infected-Susceptible) epidemic dynamics (also known as the contact process) on populations modelled by homogeneous Poisson point processes of the Euclidean plane, where the infection rate of a susceptible individual is proportional to the number of infected individuals in a disc around it. The main focus of the paper is a model where points are also subject to some random motion. Conservation equations for moment measures are leveraged to analyze the stationary regime of the point processes of infected and susceptible individuals. A heuristic factorization of the third moment measure is then proposed to obtain simple polynomial equations allowing one to derive closed form approximations for the fraction of infected individuals in the steady state. These polynomial equations also lead to a phase diagram which tentatively delineates the regions of the space of parameters (population density, infection radius, infection and recovery rate, and motion rate) where the epidemic survives and those where there is extinction. A key take-away from this phase diagram is that the extinction of the epidemic is not always aided by a decrease in the motion rate. These results are substantiated by simulations on large two dimensional tori. These simulations show that the polynomial equations accurately predict the fraction of infected individuals when the epidemic survives. The simulations also show that the proposed phase diagram accurately predicts the parameter regions where the mean survival time of the epidemic increases (resp. decreases) with motion rate.

     

State-space modelling reveals proximate causes of harbour seal population declines

Declines in large vertebrate populations are widespread but difficult to detect from monitoring data and hard to understand due to a multiplicity of plausible biological explanations. In parts of Scotland, harbour seals (Phoca vitulina) have been in decline for 10 years. To evaluate the contributions of different proximate causes (survival, fecundity, observation artefacts) to this decline, we collated behavioural, demographic and population data from one intensively studied population in part of the Moray Firth (north-east Scotland). To these, we fit a state-space model comprising age-structured dynamics and a detailed account of observation errors. After accounting for culling (estimated by our model as 14 % of total mortality), the main driver of the historical population decline was a decreasing trend in survival of young individuals combined with (previously unrecognised) low levels of pupping success. In more recent years, the model provides evidence for considerable increases in breeding success and consistently high levels of adult survival. However, breeding success remains the most volatile demographic component of the population. Forecasts from the model indicate a slow population recovery, providing cautious support for recent management measures. Such investigations of the proximate causes of population change (survival, fecundity and observation errors) provide valuable short-term support for the management of population declines, helping to focus future data collection on those ultimate causal mechanisms that are not excluded by the demographic evidence. The contribution of specific ultimate drivers (e.g. shooting mortality or competitors) can also be quantified by including them as covariates to survival or fecundity.     

Nicotine exploits a COPI-mediated process for chaperone-mediated up-regulation of its receptors

Chronic exposure to nicotine up-regulates high sensitivity nicotinic acetylcholine receptors (nAChRs) in the brain. This up-regulation partially underlies addiction and may also contribute to protection against Parkinson’s disease. nAChRs containing the α6 subunit (α6* nAChRs) are expressed in neurons in several brain regions, but comparatively little is known about the effect of chronic nicotine on these nAChRs. We report here that nicotine up-regulates α6* nAChRs in several mouse brain regions (substantia nigra pars compacta, ventral tegmental area, medial habenula, and superior colliculus) and in neuroblastoma 2a cells. We present evidence that a coat protein complex I (COPI)-mediated process mediates this up-regulation of α6* or α4* nAChRs but does not participate in basal trafficking. We show that α6β2β3 nAChR up-regulation is prevented by mutating a putative COPI-binding motif in the β3 subunit or by inhibiting COPI. Similarly, a COPI-dependent process is required for up-regulation of α4β2 nAChRs by chronic nicotine but not for basal trafficking. Mutation of the putative COPI-binding motif or inhibition of COPI also results in reduced normalized Förster resonance energy transfer between α6β2β3 nAChRs and εCOP subunits. The discovery that nicotine exploits a COPI-dependent process to chaperone high sensitivity nAChRs is novel and suggests that this may be a common mechanism in the up-regulation of nAChRs in response to chronic nicotine.     

Accounting for Biomass Carbon Stock Change Due to Wildfire in Temperate Forest Landscapes in Australia

Carbon stock change due to forest management and disturbance must be accounted for in UNFCCC national inventory reports and for signatories to the Kyoto Protocol. Impacts of disturbance on greenhouse gas (GHG) inventories are important for many countries with large forest estates prone to wildfires. Our objective was to measure changes in carbon stocks due to short-term combustion and to simulate longer-term carbon stock dynamics resulting from redistribution among biomass components following wildfire. We studied the impacts of a wildfire in 2009 that burnt temperate forest of tall, wet eucalypts in south-eastern Australia. Biomass combusted ranged from 40 to 58 tC ha⁻¹, which represented 6–7% and 9–14% in low- and high-severity fire, respectively, of the pre-fire total biomass carbon stock. Pre-fire total stock ranged from 400 to 1040 tC ha⁻¹ depending on forest age and disturbance history. An estimated 3.9 TgC was emitted from the 2009 fire within the forest region, representing 8.5% of total biomass carbon stock across the landscape. Carbon losses from combustion were large over hours to days during the wildfire, but from an ecosystem dynamics perspective, the proportion of total carbon stock combusted was relatively small. Furthermore, more than half the stock losses from combustion were derived from biomass components with short lifetimes. Most biomass remained on-site, although redistributed from living to dead components. Decomposition of these components and new regeneration constituted the greatest changes in carbon stocks over ensuing decades. A critical issue for carbon accounting policy arises because the timeframes of ecological processes of carbon stock change are longer than the periods for reporting GHG inventories for national emissions reductions targets. Carbon accounts should be comprehensive of all stock changes, but reporting against targets should be based on human-induced changes in carbon stocks to incentivise mitigation activities.     

A tool for simulating and communicating uncertainty when modelling species distributions under future climates

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