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

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.

Mechanobiologically optimized 3D titanium-mesh scaffolds enhance bone regeneration in critical segmental defects in sheep.

Three-dimensional (3D) titanium-mesh scaffolds offer many advantages over autologous bone grafting for the regeneration of challenging large segmental bone defects. Our study supports the hypothesis that endogenous bone defect regeneration can be promoted by mechanobiologically optimized Ti-mesh scaffolds. Using finite element techniques, two mechanically distinct Ti-mesh scaffolds were designed in a honeycomb-like configuration to minimize stress shielding while ensuring resistance against mechanical failure. Scaffold stiffness was altered through small changes in the strut diameter only. Honeycombs were aligned to form three differently oriented channels (axial, perpendicular, and tilted) to guide the bone regeneration process. The soft scaffold (0.84 GPa stiffness) and a 3.5-fold stiffer scaffold (2.88 GPa) were tested in a critical size bone defect model in vivo in sheep. To verify that local scaffold stiffness could enhance healing, defects were stabilized with either a common locking compression plate that allowed dynamic loading of the 4-cm defect or a rigid custom-made plate that mechanically shielded the defect. Lower stress shielding led to earlier defect bridging, increased endochondral bone formation, and advanced bony regeneration of the critical size defect. This study demonstrates that mechanobiological optimization of 3D additive manufactured Ti-mesh scaffolds can enhance bone regeneration in a translational large animal study.

Botulinum toxin type A treatment to the upper face: retrospective analysis of daily practice.

BACKGROUND: Botulinum toxin type A treatment has been used for over 20 years to enhance the appearance of the face. There are several commercially available botulinum toxin type A products used in aesthetic clinical practice. The aim of this retrospective analysis was to compare the clinical efficacy of the most commonly used botulinum toxin type A preparations in daily practice. METHODS: Physicians from 21 centers in Germany completed questionnaires based on an inspection of subject files for subjects 18 years of age or over who had received at least two, but not more than three, consecutive treatments with incobotulinumtoxinA, onabotulinumtoxinA, or abobotulinumtoxinA within a 12-month period in the previous 2 years. Data on subject and physician satisfaction, treatment intervals, dosages, and safety were collected from 1256 subjects. RESULTS: There were no statistically significant differences between incobotulinumtoxinA and onabotulinumtoxinA with respect to physician and subject satisfaction, dosages, and adverse effects experienced. Both botulinum toxin type A preparations were well tolerated and effective in the treatment of upper facial lines. Due to low treatment numbers, abobotulinumtoxinA was not included in the statistical analysis. CONCLUSION: The results of this retrospective analysis confirm the results of prospective clinical trials by demonstrating that, in daily practice, incobotulinumtoxinA and onabotulinumtoxinA are used at a 1:1 dose ratio and display comparable efficacy and safety.

Quantitative determination of estrogen receptor, progesterone receptor, and HER2 mRNA in formalin-fixed paraffin-embedded tissue--a new option for predictive biomarker assessment in breast cancer.

The development of optimized therapy strategies against malignant tumors is critically dependent on the assessment of tissue-based biomarkers in routine diagnostic tissue samples. We investigated a novel, fully automated, and xylene-free method for RNA isolation and biomarker determination using formalin-fixed paraffin-embedded (FFPE) tissue. The aim was to show that this approach is feasible and gives results that are comparable to the current gold standards. Expression of the breast cancer biomarkers ESR1, PGR, and HER2 was measured in a total of 501 FFPE tissue samples from 167 breast carcinomas, which had been stored for up to 21 years. Total RNA was extracted from tissue sections and biomarker expression was measured by kinetic RT-PCR (RT-kPCR). The results of the new method were compared with immunohistochemistry as the current gold standard.RNA was successfully isolated from all samples, with a mean yield of 1.4 µg/sample and fragment lengths of at least 150 bp in 99% of samples. RT-kPCR analysis of ESR1, PGR, and HER2 was possible in all samples. Comparing RT-kPCR results with standard IHC, we found a good concordance for ESR1 (agreement: 98.4%), PGR (84.4%), and HER2 (89.8%). We observed a low section-to-section variability of kPCR results for all 3 biomarkers (root of mean squared errors: 0.2 to 0.5 Ct values). The new approach is a reliable high-throughput instrument for standardized testing of biomarkers in clinical routine and for research studies on archived FFPE material up to 21 years old. For the assessment of ESR1, PGR, and HER2 the results are comparable to the current gold-standard.

Comparison of microarray-based RNA expression with ELISA-based protein determination of HER2, uPA and PAI-1 in tumour tissue of patients with breast cancer and relation to outcome.

PURPOSE: Prognostic and predictive markers in breast cancer are currently determined by single analysis of protein amounts. If RNA-based multi-gene analyses enter clinical practice, simultaneous determination of currently established markers like human epidermal growth factor receptor 2 (HER2), urokinase plasminogen activator (uPA) and its inhibitor (PAI-1) would represent an elegant simplification. To investigate the correlation between RNA and protein levels, we assessed HER2, uPA and PAI-1 in patients with breast cancer. In addition, we evaluated the influence of these factors on patient outcome. METHODS: We collected tumour samples from 133 patients with primary breast cancer. Protein and mRNA levels were measured for HER2, uPA and PAI-1. Protein concentration was measured by ELISA, mRNA expression was analysed by Affymetrix A133U Gene Chip and validated by quantitative PCR. RESULTS: We were able to demonstrate a statistically significant correlation between mRNA and protein expression for HER2 (r = 0.67, P < 0.001) and uPA (r = 0.7, P < 0.001) but not for PAI-1 (r = 0.27). We observed a prognostic information for PAI-1 mRNA and protein values. Patients with high PAI-1 mRNA expression had a reduced 10-year disease-free survival (DFS) rate (60 vs. 70%, P = 0.071) and 10-year overall survival (OS) rate (68 vs. 79%, P = 0.034). Patients with PAI-1 protein levels above 14 ng/mg protein had a reduced disease-free (10-year DFS rate 54 vs. 71%, P = 0.006) and overall survival rate (10-year OS-rate 63 vs. 83%, P = 0.018). In the patient cohort with no chemotherapy, PAI-1 mRNA levels were the strongest prognostic factor for OS in univariate and multivariate analysis. CONCLUSIONS: Results of RNA-based multi-gene analyses of the prognostic and predictive markers HER2 and uPA correlate with the corresponding protein levels. This is not the case for PAI-1. However, PAI-1 mRNA expression might reveal new clinically relevant information in addition to PAI protein levels.