Dendrogram showing hierarchical clustering of patients according to the exploratory miRNA screening panel results.

<p>Young patients are shown in black, older patients are shown in grey. The clustering was performed on all 20 patients of the exploratory cohort and on the entire miRNA screening panel, applying complete linkage as the clustering method and Euclidean distance as the distance measure.</p

Patient and tumor characteristics of the pilot study cohort.

<p>Patient and tumor characteristics of the pilot study cohort.</p

Principal component analysis (PCA) of the panel screening results.

<p>(<b>A</b>) PCA analysis of the top-50 ranking (lowest P-values in t-test) miRNAs of the panel screening; (<b>B</b>) PCA analysis of the bottom-50 ranking miRNAs (highest P-values in t-test). Black symbols represent young patients; grey symbols represent older, frail patients. PC1, PC2 and PC3 on X, Y and Z-axis represent the 3 principal components generated by this statistical algorithm.</p

Validation of differentially expressed miRNAs in older (N = 10) <i>versus</i> young (N = 20) breast cancer patients and comparison between older ‘fit’ (N = 10) and older ‘frail’ (N = 10) subjects.

a<p>P-values are derived from parametric t-test (unpaired, 2-tail, C.I = 95%), unless data are not normally distributed (values indicated <i>in italic</i>). In such cases, P-value derived from Mann-Whitney test is displayed. <b>Bold values</b> indicate significant values according to the 5% significance threshold. Values indicated with an asterisk (<b>*</b>) remained significant after correction for multiple testing.</p>b<p>Negative values indicate x-fold <i>decreased</i> expression, positive values indicate x-fold <i>increased</i> expression.</p><p>Validation of differentially expressed miRNAs in older (N = 10) <i>versus</i> young (N = 20) breast cancer patients and comparison between older ‘fit’ (N = 10) and older ‘frail’ (N = 10) subjects.</p

Candidate reference miRNAs identified by exploratory serum/plasma miRNA panel screening.

a<p>Variance after global mean normalisation (GMN) : reflects the deviation of a specific miRNA from the behavior of the global mean (i.e. average signal of all miRNAs included).</p>b<p>Standard deviation returned by NormFinder algorithm.</p>c<p>Expression stability measure (M) returned by GeNorm algorithm (the lower, the more stable the expression).</p><p>Candidate reference miRNAs identified by exploratory serum/plasma miRNA panel screening.</p

Patient and tumor characteristics of the validation study cohort.

a<p>Leuven Oncology Frailty Score : refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110644#pone.0110644.s001" target="_blank">File S1</a> for more details.</p>b<p>N/A : not applicable.</p>c<p>One patient had a mixed luminal A (grade II)/luminal B (grade III) tumor.</p><p>Patient and tumor characteristics of the validation study cohort.</p

Experimental study design.

<p>Experimental study design.</p

Venn diagram showing up-regulated and down-regulated miRNAs in the exploratory screening and the validation study.

<p>In the exploratory screening, there were in total 37 circulating miRNAs exhibiting age-related expression (p<0.05). Of those, only the top-15 miRNAs with the lowest p-values (shown in grey boxes) were further investigated in the validation study. Seven age-related miRNAs were confirmed in the validation study (p<0.05), of which 5 (all showing decreased expression) remained significant after correction for multiple testing; these are indicated in bold. Obviously, the right-hand zone (i.e. miRNAs showing altered expression in the validation study but not the exploratory screening) cannot contain any miRNAs, since the validation study only included those miRNAs that emerged from the exploratory study as the most significantly age-related ones.</p

Differential expression of the top-15 ranking miRNAs from the panel screening.

<p>The selected miRNAs are displayed in numerical order. Bars represent the difference in expression in older compared to young patients on a 2-logarithmic scale, with indication of the 95% confidence interval (based on t-test).</p

Example of manual and semi-automated images evaluation.

<p>This figure shows a case where 3D measurements could detect a size reduction >20%, and 1D measurements did not, because the shrinkage was mostly in the shortest diameters of the target lesion. To note, the shortest diameter (not taken into account by RECIST) decreased of 20% and 22% at 6 and 12 months respectively. Images are shown in the axial plane and in the coronal plan, and 3D reconstruction with OsiriX® Imaging Software is shown in the upper, middle, and lower part of the figure, respectively. The number of CT slices where the lesion could be identified at different time-points is indicated. Interestingly, this lesion was classified as SD according to RECIST and 3D-sphere criteria, and as PR according to 3D-ellipsoid and Choi criteria at both time-points.</p