Reverse transcription-quantitative polymerase chain reaction: description of a RIN-based algorithm for accurate data normalization

<p>Abstract</p> <p>Background</p> <p>Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the gold standard technique for mRNA quantification, but appropriate normalization is required to obtain reliable data. Normalization to accurately quantitated RNA has been proposed as the most reliable method for in vivo biopsies. However, this approach does not correct differences in RNA integrity.</p> <p>Results</p> <p>In this study, we evaluated the effect of RNA degradation on the quantification of the relative expression of nine genes (<it>18S</it>, <it>ACTB</it>, <it>ATUB</it>, <it>B2M</it>, <it>GAPDH</it>, <it>HPRT</it>, <it>POLR2L</it>, <it>PSMB6</it> and <it>RPLP0</it>) that cover a wide expression spectrum. Our results show that RNA degradation could introduce up to 100% error in gene expression measurements when RT-qPCR data were normalized to total RNA. To achieve greater resolution of small differences in transcript levels in degraded samples, we improved this normalization method by developing a corrective algorithm that compensates for the loss of RNA integrity. This approach allowed us to achieve higher accuracy, since the average error for quantitative measurements was reduced to 8%. Finally, we applied our normalization strategy to the quantification of <it>EGFR</it>, <it>HER2 </it>and <it>HER3 </it>in 104 rectal cancer biopsies. Taken together, our data show that normalization of gene expression measurements by taking into account also RNA degradation allows much more reliable sample comparison.</p> <p>Conclusion</p> <p>We developed a new normalization method of RT-qPCR data that compensates for loss of RNA integrity and therefore allows accurate gene expression quantification in human biopsies.</p

Short-Term cost impact of compliance with clinical practice guidelines for initial sarcoma treatment

Background: The impact of compliance to clinical practice guidelines (CPG) on outcomes and/or costs of care has not been completely clarified.Objective: To estimate relationships between medical expenditures and compliance to CPG for initial sarcoma treatment.Research design: Selected cohorts of patients diagnosed with sarcoma in 2005 and 2006, and treated at the University hospital and/or the cancer centre of the Rhône-Alpes region, France (n=90). Main outcome measurements were: patient characteristics, compliance with CPG, health outcomes, and costs. Data were mainly extracted from patient records. The logarithm of treatment costs was modelled using linear and Tobit regressions.Results: Rates of compliance with CPG were 86%, 66%, 88%, 89%, and 95% for initial diagnosis, primary surgical excision, wide surgical excision, chemotherapy, and radiotherapy, respectively. Total average costs reached €24,439, with €1,784, €11,225, €10,360, and €1,016 for diagnosis, surgery (primary and wide surgical excisions), chemotherapy, and radiotherapy, respectively. Compliance of diagnosis with CPG decreased the cost of diagnosis, whereas compliance of primary surgical excision increased the cost of chemotherapy. Compliance of chemotherapy with CPG decreased the cost of radiotherapy.Conclusion: Since chemotherapy is one of the major cost drivers, these results support that compliance with guidelines increases medical care expenditures in short term.Oncology; Sarcoma; Cost; Clinical guidelines; Efficacy; Medical Practices; Government Policy; Regulation; Public Health

Minimum Information About a Simulation Experiment (MIASE)

The original publication is available at www.ploscompbiol.orgReproducibility of experiments is a basic requirement for science. Minimum Information (MI) guidelines have proved a helpful means of enabling reuse of existing work in modern biology. The Minimum Information Required in the Annotation of Models (MIRIAM) guidelines promote the exchange and reuse of biochemical computational models. However, information about a model alone is not sufficient to enable its efficient reuse in a computational setting. Advanced numerical algorithms and complex modeling workflows used in modern computational biology make reproduction of simulations difficult. It is therefore essential to define the core information necessary to perform simulations of those models. The Minimum Information About a Simulation Experiment describes the minimal set of information that must be provided to make the description of a simulation experiment available to others. It includes the list of models to use and their modifications, all the simulation procedures to apply and in which order, the processing of the raw numerical results, and the description of the final output. MIASE allows for the reproduction of any simulation experiment. The provision of this information, along with a set of required models, guarantees that the simulation experiment represents the intention of the original authors. Following MIASE guidelines will thus improve the quality of scientific reporting, and will also allow collaborative, more distributed efforts in computational modeling and simulation of biological processes.The discussions that led to the definition of MIASE benefited from the support of a Japan Partnering Award by the UK Biotechnology and Biological Sciences Research Council. DW was supported by the Marie Curie program and by the German Research Association (DFG Research Training School ‘‘dIEM oSiRiS’’ 1387/1). This publication is based on work (EJC) supported in part by Award No KUK-C1-013-04, made by King Abdullah University of Science and Technology (KAUST). FTB acknowledges support by the NIH (grant 1R01GM081070- 01). JC is supported by the European Commission, DG Information Society, through the Seventh Framework Programme of Information and Communication Technologies, under the VPH NoE project (grant number 223920). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Publishers versio

Incidence of Sarcoma Histotypes and Molecular Subtypes in a Prospective Epidemiological Study with Central Pathology Review and Molecular Testing

International audienceBACKGROUND: The exact overall incidence of sarcoma and sarcoma subtypes is not known. The objective of the present population-based study was to determine this incidence in a European region (Rhone-Alpes) of six million inhabitants, based on a central pathological review of the cases. METHODOLOGY/PRINCIPAL FINDINGS: From March 2005 to February 2007, pathology reports and tumor blocks were prospectively collected from the 158 pathologists of the Rhone-Alpes region. All diagnosed or suspected cases of sarcoma were collected, reviewed centrally, examined for molecular alterations and classified according to the 2002 World Health Organization classification. Of the 1287 patients screened during the study period, 748 met the criteria for inclusion in the study. The overall crude and world age-standardized incidence rates were respectively 6.2 and 4.8 per 100,000/year. Incidence rates for soft tissue, visceral and bone sarcomas were respectively 3.6, 2.0 and 0.6 per 100,000. The most frequent histological subtypes were gastrointestinal stromal tumor (18%; 1.1/100,000), unclassified sarcoma (16%; 1/100,000), liposarcoma (15%; 0.9/100,000) and leiomyosarcoma (11%; 0.7/100,000). CONCLUSIONS/SIGNIFICANCE: The observed incidence of sarcomas was higher than expected. This study is the first detailed investigation of the crude incidence of histological and molecular subtypes of sarcomas

Personalized Medicine: how to Switch from the Concept to the Integration into the Clinical Development Plan to Obtain Marketing Authorization

One of the challenges of the coming years is to personalize medicine in order to provide each patient with an individualized treatment plan. The three objectives of personalized medicine are to refine diagnosis, rationalize treatment and engage patients in a preventive approach. Personalization can be characterized by various descriptors whether related to the field, biology, imaging, type of lesion of the entity to be treated, comorbidity factors, coprescriptions or the environment As part of personalized medicine focused on biological markers including genetics or genomics, the integration of the clinical development plan to obtain marketing authorization may be segmented in 3 stages with a known descriptor identified before clinical development, a known descriptor discovered during clinical development or a known descriptor known after clinical development. For each stage, it is important to clearly define the technical optimization elements, to specify the expectations and objectives, to examine the methodological aspects of each clinical development phase and finally to consider the fast changing regulatory requirements in view of the few registered therapeutics complying with the definition of personalized medicine as well as the significant technological breakthroughs according to the screened and selected biomarkers. These considerations should be integrated in view of the time required for clinical development from early phase to MA, i.e. more than 10 years. Moreover, business models related to the economic environment should be taken into account when deciding whether or not to retain a biomarker allowing the selection of target populations in a general population

La médecine personnalisée : comment passer du concept à l’intégration dans un plan de développement clinique en vue d’une AMM ?

La personnalisation de la médecine est un des enjeux des prochaines années afin qu’à chaque patient corresponde une prise en charge individualisée. La médecine personnalisée a pour triple objectif : affiner le diagnostic, rationaliser la prise en charge thérapeutique et engager le patient dans une démarche préventive. La personnalisation peut être caractérisée par différents descripteurs qu’ils soient liés au terrain, à la biologie, l’imagerie, au caractère lésionnel de l’entité à traiter, aux facteurs de comorbidité, aux coprescriptions, ou à l’environnement. Dans le cadre d’une médecine personnalisée centrée sur les marqueurs biologiques incluant la génétique ou la génomique l’intégration du plan de développement clinique en vue d’une autorisation de mise sur le marché (AMM) peut être segmenté en 3 temps avec un descripteur identifié avant le développement clinique, un descripteur découvert pendant le développement clinique ou un descripteur connu après le développement clinique. Pour chacun des temps il est important de bien définir les éléments d’optimisation technique, de préciser les attentes et objectifs, de regarder les aspects méthodologiques de chacune des phases du développement clinique et enfin tenir compte des aspects règlementaires très évolutifs compte tenu du peu de thérapeutiques disposant d’une AMM répondant à la définition de la médecine personnalisée ainsi que des sauts technologiques importants selon les biomarqueurs retenus et sélectionnés. Ces considérations sont à intégrer compte tenu du temps de développement clinique en vue d’une AMM supérieur à 10 ans entre la phase précoce et l’AMM. Par ailleurs les modèles économiques liés au contexte économique sont à prendre en compte dans les choix pour retenir ou non un biomarqueur permettant de sélectionner des populations cibles dans une population générale

[Considerations for normalisation of RT-qPCR in oncology]

International audienceGene expression analysis has many applications in the management of cancer, including diagnosis, prognosis, and therapeutic care. In this context, the reverse transcription quantitative polymerase chain reaction (RT-qPCR) has become the "gold standard" for mRNA quantification. However, this technique involves several critical steps such as RNA extraction, cDNA synthesis, quantitative PCR, and analysis, which all can be source of variation. To obtain biologically meaningful results, data normalisation is required to correct sample-to-sample variations that may be introduced during this multistage process. Normalisation can be carried out against a housekeeping gene, total RNA mass, or cell number. Careful choice of the normalization method is crucial, as any variation in the reference will introduce errors in the quantification of mRNA transcripts. By reviewing the different methods available and their related problems, the aim of this article is to provide recommendations for the set up of an appropriate normalisation strategy for RT-qPCR data in oncology

Validation of an appropriate reference gene for normalization of reverse transcription-quantitative polymerase chain reaction data from rectal cancer biopsies.: Normalization of RT-qPCR data in rectal cancer

International audienceGene expression quantification using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) requires data normalization using an invariable reference gene. Here we assessed the stability of 15 housekeeping genes in 31 tumor and normal rectal samples to validate a reliable reference gene for rectal cancer studies. Our data show that 18S and 28S RNA are stably expressed in all samples. Moreover, when used for normalization, 18S, but not 28S, greatly reduced unspecific variations of gene expression due to RNA degradation. These results demonstrate that 18S is an appropriate reference gene for normalization of RT-qPCR data from rectal cancer samples

Cyclin D1 gene G870A polymorphism predicts response to neoadjuvant radiotherapy and prognosis in rectal cancer.

PURPOSE: To investigate whether CCND1 genetic variations associated with a constitutive nuclear protein may influence either the pathologic response to preoperative RT or the prognosis in a series of rectal cancer patients. METHODS AND MATERIALS: Seventy rectal cancer patients treated by neoadjuvant radiotherapy were included in the study. CCND1 exon 5 mutations were screened, and the G870A polymorphism was assessed for correlation with clinical variables, tumor response, and patient outcome. RESULTS: No exon 5 mutation was found. Concerning the G870A polymorphism, the A/A variant was significantly associated with radiosensitivity (p = 0.022). Moreover, patients harboring the A allele were correlated with a lower risk of local failure (p = 0.017). Also, combination of the G870A polymorphism with the post-therapeutic lymph node status allowed the elaboration of a prognostic index, which accurately distinguished subgroups of patients with predictable recurrence-free (p = 0.003) and overall (p = 0.044) survival. CONCLUSIONS: Although CCND1 exon 5 mutations are rare in rectal cancer, G870A polymorphism is a frequent variation that may predict radiosensitivity and prognosis

Epidemiological evaluation of concordance between initial diagnosis and central pathology review in a comprehensive and prospective series of sarcoma patients in the Rhone-Alpes region.

International audienceBACKGROUND: Sarcomas are rare malignant tumors. Accurate initial histological diagnosis is essential for adequate management. We prospectively assessed the medical management of all patients diagnosed with sarcoma in a European region over a one-year period to identify the quantity of first diagnosis compared to central expert review (CER). METHODS: Histological data of all patients diagnosed with sarcoma in Rhone-Alpes between March 2005 and Feb 2006 were collected. Primary diagnoses were systematically compared with second opinion from regional and national experts. RESULTS: Of 448 patients included, 366 (82%) matched the inclusion criteria and were analyzed. Of these, 199 (54%) had full concordance between primary diagnosis and second opinion (the first pathologist and the expert reached identical conclusions), 97 (27%) had partial concordance (identical diagnosis of conjonctive tumor but different grade or subtype), and 70 (19%) had complete discordance (different histological type or invalidation of the diagnosis of sarcoma). The major discrepancies were related to histological grade (n = 68, 19%), histological type (n = 39, 11%), subtype (n = 17, 5%), and grade plus subtype or grade plus histological type (n = 43, 12%). CONCLUSIONS: Over 45% of first histological diagnoses were modified at second reading, possibly resulting in different treatment decisions. Systematic second expert opinion improves the quality of diagnosis and possibly the management of patients