Preanalytical variables and performance of diagnostic RNA-based gene expression analysis in breast cancer

Prognostic multigene expression assays have become widely available to provide additional information to standard clinical parameters and to support clinicians in treatment decisions. In this study, we analyzed the impact of variations in tissue handling on the diagnostic EndoPredict test results. EndoPredict is a quantitative reverse transcription PCR assay conducted on RNA from formalin-fixed, paraffin-embedded (FFPE) tissue that predicts the likelihood of distant recurrence in patients with ER-positive/HER2-negative breast cancer. In this study, we performed a total of 138 EndoPredict assays to study the effects of preanalytical variables such as time to fixation, fixation time, tumor cell content, and section storage time on the EndoPredict test results. A time to fixation of up to 12 h and fixation of up to 5 days did not affect the results of the gene expression test. Paired samples of FFPE sections with tumor cell content ranging from 15 to 95 % and tumor-enriched samples showed a correlation coefficient of 0.97. Test results of tissue sections that have been stored for 12 months at +4 or +20 °C showed a correlation of 0.99 when compared to results of nonstored sections. In conclusion, preanalytical tissue handling is not a critical factor for diagnostic gene expression analysis with the EndoPredict assay. The test can therefore be easily integrated into the standard workflow of molecular pathology

Identifying good practices for detecting inter-regional linear functional connectivity from EEG

Aggregating voxel-level statistical dependencies between multivariate time series is an important intermediate step when characterising functional connectivity (FC) between larger brain regions. However, there are numerous ways in which voxel-level data can be aggregated into inter-regional FC, and the advantages of each of these approaches are currently unclear.In this study we generate ground-truth data and compare the performances of various pipelines that estimate directed and undirected linear phase-to-phase FC between regions. We test the ability of several existing and novel FC analysis pipelines to identify the true regions within which connectivity was simulated. We test various inverse modelling algorithms, strategies to aggregate time series within regions, and connectivity metrics. Furthermore, we investigate the influence of the number of interactions, the signal-to-noise ratio, the noise mix, the interaction time delay, and the number of active sources per region on the ability of detecting phase-to-phase FC.Throughout all simulated scenarios, lowest performance is obtained with pipelines involving the absolute value of coherency. Further, the combination of dynamic imaging of coherent sources (DICS) beamforming with directed FC metrics that aggregate information across multiple frequencies leads to unsatisfactory results. Pipelines that show promising results with our simulated pseudo-EEG data involve the following steps: (1) Source projection using the linearly-constrained minimum variance (LCMV) beamformer. (2) Principal component analysis (PCA) using the same fixed number of components within every region. (3) Calculation of the multivariate interaction measure (MIM) for every region pair to assess undirected phase-to-phase FC, or calculation of time-reversed Granger Causality (TRGC) to assess directed phase-to-phase FC. We formulate recommendations based on these results that may increase the validity of future experimental connectivity studies.We further introduce the free ROIconnect plugin for the EEGLAB toolbox that includes the recommended methods and pipelines that are presented here. We show an exemplary application of the best performing pipeline to the analysis of EEG data recorded during motor imagery

Gait Event Prediction Using Surface Electromyography in Parkinsonian Patients

Gait disturbances are common manifestations of Parkinson’s disease (PD), with unmet therapeutic needs. Inertial measurement units (IMUs) are capable of monitoring gait, but they lack neurophysiological information that may be crucial for studying gait disturbances in these patients. Here, we present a machine learning approach to approximate IMU angular velocity profiles and subsequently gait events using electromyographic (EMG) channels during overground walking in patients with PD. We recorded six parkinsonian patients while they walked for at least three minutes. Patient-agnostic regression models were trained on temporally embedded EMG time series of different combinations of up to five leg muscles bilaterally (i.e., tibialis anterior, soleus, gastrocnemius medialis, gastrocnemius lateralis, and vastus lateralis). Gait events could be detected with high temporal precision (median displacement of <50 ms), low numbers of missed events (<2%), and next to no false-positive event detections (<0.1%). Swing and stance phases could thus be determined with high fidelity (median F1-score of ~0.9). Interestingly, the best performance was obtained using as few as two EMG probes placed on the left and right vastus lateralis. Our results demonstrate the practical utility of the proposed EMG-based system for gait event prediction, which allows the simultaneous acquisition of an electromyographic signal to be performed. This gait analysis approach has the potential to make additional measurement devices such as IMUs and force plates less essential, thereby reducing financial and preparation overheads and discomfort factors in gait studies

Decentral gene expression analysis: analytical validation of the Endopredict genomic multianalyte breast cancer prognosis test

Abstract Background EndoPredict (EP) is a clinically validated multianalyte gene expression test to predict distant metastasis in ER-positive, HER2-negative breast cancer treated with endocrine therapy alone. The test is based on the combined analysis of 12 genes in formalin-fixed, paraffin-embedded (FFPE) tissue by reverse transcription-quantitative real-time PCR (RT-qPCR). Recently, it was shown that EP is feasible for reliable decentralized assessment of gene expression. The aim of this study was the analytical validation of the performance characteristics of the assay and its verification in a molecular-pathological routine laboratory. Methods Gene expression values to calculate the EP score were assayed by one-step RT-qPCR using RNA from FFPE tumor tissue. Limit of blank, limit of detection, linear range, and PCR efficiency were assessed for each of the 12 PCR assays using serial samples dilutions. Different breast cancer samples were used to evaluate RNA input range, precision and inter-laboratory variability. Results PCR assays were linear up to Cq values between 35.1 and 37.2. Amplification efficiencies ranged from 75% to 101%. The RNA input range without considerable change of the EP score was between 0.16 and 18.5 ng/μl. Analysis of precision (variation of day, day time, instrument, operator, reagent lots) resulted in a total noise (standard deviation) of 0.16 EP score units on a scale from 0 to 15. The major part of the total noise (SD 0.14) was caused by the replicate-to-replicate noise of the PCR assays (repeatability) and was not associated with different operating conditions (reproducibility). Performance characteristics established in the manufacturer’s laboratory were verified in a routine molecular pathology laboratory. Comparison of 10 tumor samples analyzed in two different laboratories showed a Pearson coefficient of 0.995 and a mean deviation of 0.15 score units. Conclusions The EP test showed reproducible performance characteristics with good precision and negligible laboratory-to-laboratory variation. This study provides further evidence that the EP test is suitable for decentralized testing in specialized molecular pathological laboratories instead of a reference laboratory. This is a unique feature and a technical advance in comparison with existing RNA-based prognostic multigene expression tests.</p