Dataset Information

Microarray Normalization Revisited for Reproducible Breast Cancer Biomarkers.

ABSTRACT: Precision medicine for breast cancer relies on biomarkers to select therapies. However, the reliability of biomarkers drawn from gene expression arrays has been questioned and calls for reassessment, in particular for large datasets. We revisit widely used data-normalization procedures and evaluate differences in outcome in order to pinpoint the most reliable reprocessing methods biomarkers can be based upon. We generated a database of 3753 breast cancer patients out of 38 studies by downloading and curating patient samples from NCBI-GEO. As gene-expression biomarkers, we select the assessment of receptor status and breast cancer subtype classification. Each normalization procedure is applied separately, and biomarkers are then evaluated for each patient. Differences between normalization pipelines are quantified as percentages of patients having outcomes different for each pipeline. Some normalization procedures lead to quite consistent biomarkers, differing only in 1-2% of patients. Other normalization procedures-some of them have been used in many clinical studies-end up with distrusting discrepancies (10% and more). A good deal of doubt regarding the reliability of microarrays may root in the haphazard application of inadequate preprocessing pipelines. Several modes of batch corrections are evaluated regarding a possible improvement of receptor prediction from gene expression versus the golden standard of immunohistochemistry. Finally, we nominate those normalization methods yielding consistent and trustable results. Adequate bioinformatics data preprocessing is key and crucial for any subsequent statistics to arrive at trustable results. We conclude with a suggestion for future bioinformatics development to further increase the reliability of cancer biomarkers.

SUBMITTER: Kenn M

PROVIDER: S-EPMC7428878 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Microarray Normalization Revisited for Reproducible Breast Cancer Biomarkers.

Kenn Michael M Cacsire Castillo-Tong Dan D Singer Christian F CF Cibena Michael M Kölbl Heinz H Schreiner Wolfgang W

BioMed research international 20200806

Precision medicine for breast cancer relies on biomarkers to select therapies. However, the reliability of biomarkers drawn from gene expression arrays has been questioned and calls for reassessment, in particular for large datasets. We revisit widely used data-normalization procedures and evaluate differences in outcome in order to pinpoint the most reliable reprocessing methods biomarkers can be based upon. We generated a database of 3753 breast cancer patients out of 38 studies by downloading ...[more]

PMID: 32832541

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Project description:MotivationIn cluster analysis, the validity of specific solutions, algorithms, and procedures present significant challenges because there is no null hypothesis to test and no 'right answer'. It has been noted that a replicable classification is not necessarily a useful one, but a useful one that characterizes some aspect of the population must be replicable. By replicable we mean reproducible across multiple samplings from the same population. Methodologists have suggested that the validity of clustering methods should be based on classifications that yield reproducible findings beyond chance levels. We used this approach to determine the performance of commonly used clustering algorithms and the degree of replicability achieved using several microarray datasets.MethodsWe considered four commonly used iterative partitioning algorithms (Self Organizing Maps (SOM), K-means, Clutsering LARge Applications (CLARA), and Fuzzy C-means) and evaluated their performances on 37 microarray datasets, with sample sizes ranging from 12 to 172. We assessed reproducibility of the clustering algorithm by measuring the strength of relationship between clustering outputs of subsamples of 37 datasets. Cluster stability was quantified using Cramer's v2 from a kXk table. Cramer's v2 is equivalent to the squared canonical correlation coefficient between two sets of nominal variables. Potential scores range from 0 to 1, with 1 denoting perfect reproducibility.ResultsAll four clustering routines show increased stability with larger sample sizes. K-means and SOM showed a gradual increase in stability with increasing sample size. CLARA and Fuzzy C-means, however, yielded low stability scores until sample sizes approached 30 and then gradually increased thereafter. Average stability never exceeded 0.55 for the four clustering routines, even at a sample size of 50. These findings suggest several plausible scenarios: (1) microarray datasets lack natural clustering structure thereby producing low stability scores on all four methods; (2) the algorithms studied do not produce reliable results and/or (3) sample sizes typically used in microarray research may be too small to support derivation of reliable clustering results. Further research should be directed towards evaluating stability performances of more clustering algorithms on more datasets specially having larger sample sizes with larger numbers of clusters considered.

Project description:BackgroundAt the molecular level breast cancer comprises a heterogeneous set of subtypes associated with clear differences in gene expression and clinical outcomes. Single sample predictors (SSPs) are built via a two-stage approach consisting of clustering and subtype predictor construction based on the cluster labels of individual cases. SSPs have been criticized because their subtype assignments for the same samples were only moderately concordant (Cohen's κ<0.6).MethodsWe propose a semi-supervised approach where for five datasets, consensus sets were constructed consisting of those samples that were concordantly subtyped by a number of different predictors. Next, nine subtype predictors - three SSPs, three subtype classification models (SCMs) and three novel rule-based predictors based on the St. Gallen surrogate intrinsic subtype definitions (STGs) - were constructed on the five consensus sets and their associated consensus subtype labels. The predictors were validated on a compendium of over 4,000 uniformly preprocessed Affymetrix microarrays. Concordance between subtype predictors was assessed using Cohen's kappa statistic.ResultsIn this standardized setup, subtype predictors of the same type (either SCM, SSP, or STG) but with a different gene list and/or consensus training set were associated with almost perfect levels of agreement (median κ>0.8). Interestingly, for a given predictor type a change in consensus set led to higher concordance than a change to another gene list. The more challenging scenario where the predictor type, gene list and training set were all different resulted in predictors with only substantial levels of concordance (median κ=0.74) on independent validation data.ConclusionsOur results demonstrate that for a given subtype predictor type stringent standardization of the preprocessing stage, combined with carefully devised consensus training sets, leads to predictors that show almost perfect levels of concordance. However, predictors of a different type are only substantially concordant, despite reaching almost perfect levels of concordance on training data.

Dataset Information

Microarray Normalization Revisited for Reproducible Breast Cancer Biomarkers.

Publications

Microarray Normalization Revisited for Reproducible Breast Cancer Biomarkers.

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