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Selection of microsatellite markers for bladder cancer diagnosis without the need for corresponding blood - PubMed

Selection of microsatellite markers for bladder cancer diagnosis without the need for corresponding blood

Angela A G van Tilborg et al. PLoS One. 2012.

Abstract

Microsatellite markers are used for loss-of-heterozygosity, allelic imbalance and clonality analyses in cancers. Usually, tumor DNA is compared to corresponding normal DNA. However, normal DNA is not always available and can display aberrant allele ratios due to copy number variations in the genome. Moreover, stutter peaks may complicate the analysis. To use microsatellite markers for diagnosis of recurrent bladder cancer, we aimed to select markers without stutter peaks and a constant ratio between alleles, thereby avoiding the need for a control DNA sample. We investigated 49 microsatellite markers with tri- and tetranucleotide repeats in regions commonly lost in bladder cancer. Based on analysis of 50 blood DNAs the 12 best performing markers were selected with few stutter peaks and a constant ratio between peaks heights. Per marker upper and lower cut off values for allele ratios were determined. LOH of the markers was observed in 59/104 tumor DNAs. We then determined the sensitivity of the marker panel for detection of recurrent bladder cancer by assaying 102 urine samples of these patients. Sensitivity was 63% when patients were stratified for LOH in their primary tumors. We demonstrate that up-front selection of microsatellite markers obliterates the need for a corresponding blood sample. For diagnosis of bladder cancer recurrences in urine this significantly reduces costs. Moreover, this approach facilitates retrospective analysis of archival tumor samples for allelic imbalance.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Flow chart of the study.
Figure 2
Figure 2. Overview of the variation between allele ratios for different markers.

On the Y-axis, the ratio between the two alleles is given. On the X-axis, the different microsatellite markers are listed. A. The boxplots show that some previously used markers have a large variation in their allele ratio based on an analysis of blood DNA samples from 50 individuals. B. Behavior of the 12 selected markers, indicating they have very little variation in their allele ratio when tested on normal blood and urine from healthy individuals. C. In primary tumor DNA the allele ratio is much more variable due to LOH/AI.

Figure 3
Figure 3. Examples of the electropherograms for the selected markers, ordered to their chromosomal position.

On the Y-axis, the peak intensity is given. On the X-axis, the fragment size is given in basepairs. On the left side, results from normal tissue are shown. Note that these markers have few or no stutter peaks and a fairly constant ratio (close to 1) between the heights of the two alleles. On the right side, results from representative tumor samples with LOH are shown.

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Grants and funding

This work was supported by European Community Seventh Framework program FP7/2007-2012, grant agreement n° 201663; Dutch Cancer Society grant no. EMCR 2007-3863. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.