Unaccounted uncertainty from qPCR efficiency estimates entails uncontrolled false positive rates

BACKGROUND: Accurate adjustment for the amplification efficiency (AE) is an important part of real-time quantitative polymerase chain reaction (qPCR) experiments. The most commonly used correction strategy is to estimate the AE by dilution experiments and use this as a plug-in when efficiency correcting the ΔΔC(q). Currently, it is recommended to determine the AE with high precision as this plug-in approach does not account for the AE uncertainty, implicitly assuming an infinitely precise AE estimate. Determining the AE with such precision, however, requires tedious laboratory work and vast amounts of biological material. Violation of the assumption leads to overly optimistic standard errors of the ΔΔC(q), confidence intervals, and p-values which ultimately increase the type I error rate beyond the expected significance level. As qPCR is often used for validation it should be a high priority to account for the uncertainty of the AE estimate and thereby properly bounding the type I error rate and achieve the desired significance level. RESULTS: We suggest and benchmark different methods to obtain the standard error of the efficiency adjusted ΔΔC(q) using the statistical delta method, Monte Carlo integration, or bootstrapping. Our suggested methods are founded in a linear mixed effects model (LMM) framework, but the problem and ideas apply in all qPCR experiments. The methods and impact of the AE uncertainty are illustrated in three qPCR applications and a simulation study. In addition, we validate findings suggesting that MGST1 is differentially expressed between high and low abundance culture initiating cells in multiple myeloma and that microRNA-127 is differentially expressed between testicular and nodal lymphomas. CONCLUSIONS: We conclude, that the commonly used efficiency corrected quantities disregard the uncertainty of the AE, which can drastically impact the standard error and lead to increased false positive rates. Our suggestions show that it is possible to easily perform statistical inference of ΔΔC(q), whilst properly accounting for the AE uncertainty and better controlling the false positive rate

Double versus single high-dose melphalan 200 mg/m2 and autologous stem cell transplantation for multiple myeloma: a region-based study in 484 patients from the Nordic area

Autologous stem cell transplantation is still considered the standard of care in young patients with multiple myeloma (MM). This disease is the most common indication for high-dose therapy (HDT) supported by hematopoietic stem cell transplantation and much data support the benefit of this procedure. Results of randomized studies are in favor of tandem autologous transplantation although the effect on overall survival is unclear. Based on sequential registration trials in the Nordic area, we aimed to evaluate the outcome of conventional single or double HDT

Abnormal IGF-Binding Protein Profile in the Bone Marrow of Multiple Myeloma Patients

Insulin-like growth factor (IGF) signalling plays a key role in homing, progression, and treatment resistance in multiple myeloma (MM). In the extracellular environment, the majority of IGF molecules are bound to one of six IGF-binding proteins (IGFBP1-6), leaving a minor fraction of total IGF free and accessible for receptor activation. In MM, high IGF-receptor type 1 expression levels correlate with a poor prognosis, but the status and role of IGF and IGFBPs in the pathobiology of MM is unknown. Here we measured total IGF1, IGF2, and intact IGFBP levels in blood and bone marrow samples from MM (n = 17), monoclonal gammopathy of undetermined significance (MGUS) (n = 37), and control individuals (n = 15), using ELISA (IGFs) and 125I-IGF1 Western Ligand Blotting (IGFBPs). MGUS and MM patients displayed a significant increase in intact IGFBP-2 (2.5-3.8 fold) and decrease in intact IGFBP-3 (0.6-0.5 fold) in the circulation compared to control individuals. Further, IGFBP-2 as well as total IGFBP levels were significantly lower in bone marrow compared to circulation in MM and MGUS only, whereas IGF1, IGF2, and IGFBP-3 were equally distributed between the two compartments. In conclusion, the profound change in IGFBP profile strongly suggests an increased IGF bioavailability in the bone marrow microenvironment in MGUS and MM, despite no change in growth factor concentration

Stringent or nonstringent complete remission and prognosis in acute myeloid leukemia:a Danish population-based study

Key Points sCR and non-sCR in acute myeloid leukemia are associated with different prognoses. Three lineage blood cell recoveries have independent impacts on prognosis.</jats:p

Next Generation Nuclear Plant Methods Technical Program Plan

One of the great challenges of designing and licensing the Very High Temperature Reactor (VHTR) is to confirm that the intended VHTR analysis tools can be used confidently to make decisions and to assure all that the reactor systems are safe and meet the performance objectives of the Generation IV Program. The research and development (R&D) projects defined in the Next Generation Nuclear Plant (NGNP) Design Methods Development and Validation Program will ensure that the tools used to perform the required calculations and analyses can be trusted. The Methods R&D tasks are designed to ensure that the calculational envelope of the tools used to analyze the VHTR reactor systems encompasses, or is larger than, the operational and transient envelope of the VHTR itself. The Methods R&D focuses on the development of tools to assess the neutronic and thermal fluid behavior of the plant. The fuel behavior and fission product transport models are discussed in the Advanced Gas Reactor (AGR) program plan. Various stress analysis and mechanical design tools will also need to be developed and validated and will ultimately also be included in the Methods R&D Program Plan. The calculational envelope of the neutronics and thermal-fluids software tools intended to be used on the NGNP is defined by the scenarios and phenomena that these tools can calculate with confidence. The software tools can only be used confidently when the results they produce have been shown to be in reasonable agreement with first-principle results, thought-problems, and data that describe the “highly ranked” phenomena inherent in all operational conditions and important accident scenarios for the VHTR

Measurement of the cosmic ray spectrum above 4×10184{\times}10^{18} eV using inclined events detected with the Pierre Auger Observatory

A measurement of the cosmic-ray spectrum for energies exceeding $4{\times}10^{18}$ eV is presented, which is based on the analysis of showers with zenith angles greater than $60^{\circ}$ detected with the Pierre Auger Observatory between 1 January 2004 and 31 December 2013. The measured spectrum confirms a flux suppression at the highest energies. Above $5.3{\times}10^{18}$ eV, the "ankle", the flux can be described by a power law $E^{-\gamma}$ with index $\gamma=2.70 \pm 0.02 \,\text{(stat)} \pm 0.1\,\text{(sys)}$ followed by a smooth suppression region. For the energy ($E_\text{s}$) at which the spectral flux has fallen to one-half of its extrapolated value in the absence of suppression, we find $E_\text{s}=(5.12\pm0.25\,\text{(stat)}^{+1.0}_{-1.2}\,\text{(sys)}){\times}10^{19}$ eV.Comment: Replaced with published version. Added journal reference and DO

The differentiation status of primary gonadal germ cell tumors correlates inversely with telomerase activity and the expression level of the gene encoding the catalytic subunit of telomerase

BACKGROUND: The activity of the ribonucleoprotein enzyme telomerase is detectable in germ, stem and tumor cells. One major component of telomerase is human telomerase reverse transcriptase (hTERT), which encodes the catalytic subunit of telomerase. Here we investigate the correlation of telomerase activity and hTERT gene expression and the differentiation status of primary testicular germ cell tumors (TGCT). METHODS: Telomerase activity (TA) was detected by a quantitative telomerase PCR ELISA, and hTERT mRNA expression was quantified by online RT-PCR in 42 primary testicular germ cell tumors. The control group consisted of benign testicular biopsies from infertile patients. RESULTS: High levels of telomerase activity and hTERT expression were detected in all examined undifferentiated TGCTs and in the benign testicular tissue specimens with germ cell content. In contrast, differentiated teratomas and testicular control tissue without germ cells (Sertoli-cell-only syndrome) showed no telomerase activity and only minimal hTERT expression. CONCLUSIONS: These findings demonstrate an inverse relationship between the level of telomerase activity and hTERT mRNA expression and the differentiation state of germ cell tumors. Quantification of telomerase activity and hTERT mRNA expression enables a new molecular-diagnostic subclassification of germ cell tumors that describes their proliferation potential and differentiation status