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Converting a breast cancer microarray signature into a high-throughput diagnostic test.

ABSTRACT: BACKGROUND: A 70-gene tumor expression profile was established as a powerful predictor of disease outcome in young breast cancer patients. This profile, however, was generated on microarrays containing 25,000 60-mer oligonucleotides that are not designed for processing of many samples on a routine basis. RESULTS: To facilitate its use in a diagnostic setting, the 70-gene prognosis profile was translated into a customized microarray (MammaPrint) containing a reduced set of 1,900 probes suitable for high throughput processing. RNA of 162 patient samples from two previous studies was subjected to hybridization to this custom array to validate the prognostic value. Classification results obtained from the original analysis were then compared to those generated using the algorithms based on the custom microarray and showed an extremely high correlation of prognosis prediction between the original data and those generated using the custom mini-array (p < 0.0001). CONCLUSION: In this report we demonstrate for the first time that microarray technology can be used as a reliable diagnostic tool. The data clearly demonstrate the reproducibility and robustness of the small custom-made microarray. The array is therefore an excellent tool to predict outcome of disease in breast cancer patients.

SUBMITTER: Glas AM

PROVIDER: S-EPMC1636049 | biostudies-literature | 2006

REPOSITORIES: biostudies-literature

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Converting a breast cancer microarray signature into a high-throughput diagnostic test.

Glas Annuska M AM Floore Arno A Delahaye Leonie J M J LJ Witteveen Anke T AT Pover Rob C F RC Bakx Niels N Lahti-Domenici Jaana S T JS Bruinsma Tako J TJ Warmoes Marc O MO Bernards René R Wessels Lodewyk F A LF Van't Veer Laura J LJ

BMC genomics 20061030

<h4>Background</h4>A 70-gene tumor expression profile was established as a powerful predictor of disease outcome in young breast cancer patients. This profile, however, was generated on microarrays containing 25,000 60-mer oligonucleotides that are not designed for processing of many samples on a routine basis.<h4>Results</h4>To facilitate its use in a diagnostic setting, the 70-gene prognosis profile was translated into a customized microarray (MammaPrint) containing a reduced set of 1,900 prob ...[more]

PMID: 17074082

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Project description:IntroductionBreast cancer is the most diagnosed and second leading cause of cancer deaths in the U.S. female population. An estimated 5 to 10 percent of all breast cancers are inherited, caused by mutations in the breast cancer susceptibility genes (BRCA1/2). As many as 90% of all mutations are nonsense mutations, causing a truncated polypeptide product. A popular and low cost method of mutation detection has been the protein truncation test (PTT), where target regions of BRCA1/2 are PCR amplified, transcribed/translated in a cell-free protein synthesis system and analyzed for truncated polypeptides by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. We previously reported a novel High Throughput Solid-Phase PTT (HTS-PTT) based on an enzyme-linked immunosorbent assay (ELISA) format that eliminates the need for radioactivity, SDS-PAGE and subjective interpretation of the results. Here, we report the next generation HTS-PTT using triple-epitope-tagged proteins and demonstrate, for the first time, its efficacy on clinical genomic DNA samples for BRCA1/2 analysis.MethodsSegments of exons 11 of BRCA1/2 open reading frames were PCR amplified from either blood derived genomic DNA or cell line mRNA. PCR primers incorporate elements for cell-free transcription/translation and epitope tagging. Cell-free expressed nascent proteins are then antibody-captured onto the wells of a microtiter plate and the relative amount of truncated polypeptide measured using antibodies against the N- and C-terminal epitope tags in an ELISA format.Results100% diagnostic sensitivity and 96% specificity for truncating mutations in exons 11 of BRCA1/2 was achieved on one hundred blood-derived clinical genomic DNA samples which were previously assayed using the conventional gel based PTT. Feasibility of full gene coverage for BRCA1/2 using mRNA source material is also demonstrated.ConclusionsOverall, the HTS-PTT provides a simple, quantitative, objective, low cost and high throughput method for analysis of truncating mutations as an alternative to gel based PTT for BRCA analysis. The technology is readily accessible to virtually any laboratory, with the only major instrumentation required being a PCR thermocycler and a basic micro-well plate reader. When compared to conventional gel based PTT, the HTS-PTT provides excellent concordance.

Project description:An increasing number of studies have profiled tumor specimens using distinct microarray platforms and analysis techniques. With the accumulating amount of microarray data, one of the most intriguing yet challenging tasks is to develop robust statistical models to integrate the findings.By applying a two-stage Bayesian mixture modeling strategy, we were able to assimilate and analyze four independent microarray studies to derive an inter-study validated "meta-signature" associated with breast cancer prognosis. Combining multiple studies (n = 305 samples) on a common probability scale, we developed a 90-gene meta-signature, which strongly associated with survival in breast cancer patients. Given the set of independent studies using different microarray platforms which included spotted cDNAs, Affymetrix GeneChip, and inkjet oligonucleotides, the individually identified classifiers yielded gene sets predictive of survival in each study cohort. The study-specific gene signatures, however, had minimal overlap with each other, and performed poorly in pairwise cross-validation. The meta-signature, on the other hand, accommodated such heterogeneity and achieved comparable or better prognostic performance when compared with the individual signatures. Further by comparing to a global standardization method, the mixture model based data transformation demonstrated superior properties for data integration and provided solid basis for building classifiers at the second stage. Functional annotation revealed that genes involved in cell cycle and signal transduction activities were over-represented in the meta-signature.The mixture modeling approach unifies disparate gene expression data on a common probability scale allowing for robust, inter-study validated prognostic signatures to be obtained. With the emerging utility of microarrays for cancer prognosis, it will be important to establish paradigms to meta-analyze disparate gene expression data for prognostic signatures of potential clinical use.

Project description:Micro- and nanoscale technologies have radically transformed biological research from genomics to tissue engineering, with the relative exception of microbial cell culture, which is still largely performed in microtiter plates and petri dishes. Here, we present nanoscale culture of the opportunistic fungal pathogen Candida albicans on a microarray platform. The microarray consists of 1,200 individual cultures of 30 nl of C. albicans biofilms ("nano-biofilms") encapsulated in an inert alginate matrix. We demonstrate that these nano-biofilms are similar to conventional macroscopic biofilms in their morphological, architectural, growth, and phenotypic characteristics. We also demonstrate that the nano-biofilm microarray is a robust and efficient tool for accelerating the drug discovery process: (i) combinatorial screening against a collection of 28 antifungal compounds in the presence of immunosuppressant FK506 (tacrolimus) identified six drugs that showed synergistic antifungal activity, and (ii) screening against the NCI challenge set small-molecule library identified three heretofore-unknown hits. This cell-based microarray platform allows for miniaturization of microbial cell culture and is fully compatible with other high-throughput screening technologies.Microorganisms are typically still grown in petri dishes, test tubes, and Erlenmeyer flasks in spite of the latest advances in miniaturization that have benefitted other allied research fields, including genomics and proteomics. Culturing microorganisms in small scale can be particularly valuable in cutting down time, cost, and reagent usage. This paper describes the development, characterization, and application of nanoscale culture of an opportunistic fungal pathogen, Candida albicans. Despite a more than 2,000-fold reduction in volume, the growth characteristics and drug response profiles obtained from the nanoscale cultures were comparable to the industry standards. The platform also enabled rapid identification of new drug candidates that were effective against C. albicans biofilms, which are a major cause of mortality in hospital-acquired infections.

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Converting a breast cancer microarray signature into a high-throughput diagnostic test.

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Converting a breast cancer microarray signature into a high-throughput diagnostic test.

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