Kaplan-Meier survival curves for miR-9.

<p>Solid lines represent LR-free survival curves of breast cancer patients who had miR-9 low expression tumors in validation sample set, all cases (panel A) and ER positive cases (panel B). Dotted lines represent the patients who hadmiR-9 high expression tumors. P values are 0.08 and 0.02 for all cases (panel A) and ER positive cases (panel B), respectively.</p

Association of miR-9 and miR-375 expression levels with tumor estrogen receptor (ER) status.

<p>Delta Ct values of miR-9 (panel A) and miR-375 (panel B) are compared between patients who had ER negative (ER status  = 0) and ER positive (ER status  = 1) tumors. A high delta Ct value indicates a low expression level. The capabilities of miR-9 and miR-375 to discriminate ER status are shown in ROC curves (panel C and D, respectively).</p

Association of miR-9 and miR-375 with selected patient characteristics.

*<p><i>P</i> values were based on Wilcoxon rank sum test (continuous variables) or Fisher’s exact test (categorical variables) ∧estrogen receptor.</p

Association of miR-9 expression levels with breast cancer LR.

<p>Delta Ct values of miR-9 are compared between breast cancer patients with (recurrence status  = 1) and without (recurrence status  = 0) breast cancer LR in all tumors (panel A), and in estrogen receptor positive (panel C) and negative (panel E) tumors. A high delta Ct value indicates a low expression level. ROC curves are drawn to show the capability of miR-9 to discriminate LR in all tumors (panel B), ER positive tumors (panel D) and ER negative tumors (panel F).</p

miR candidate selection and validation.

*<p>p-values were based on Wilcoxon rank sum test.</p

Distribution of sample characteristics by patient LR status.

<p>∗p-values were based on Wilcoxon rank sum test (continuous variables), chi-square or Fisher’s exact test (categorical variables) ∧estrogen receptor.</p

Comparisons of SYK-regulated mRNA changes, average copy numbers, and percent of cases altered.

<p><b>A.</b> The % altered IDC cases were plotted for each member of the 55 Gene Set. <b>B.</b> The average copy number was plotted for each member of the 55 Gene Set . <b>C.</b> Fold changes in mRNAs (log) of genes regulating motility and invasion following SYK siRNA knockdown in ER-negative, MCF10A breast cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087610#pone.0087610-Sung1" target="_blank">[3]</a> were plotted for 51 mRNA species.</p

<i>SYK</i> Allelic Loss and the Role of Syk-Regulated Genes in Breast Cancer Survival

<div><p>Heterozygotic loss of <i>SYK</i>, a non-receptor tyrosine kinase, gives rise to mouse mammary tumor formation where Syk protein levels are reduced by about half; loss of <i>SYK</i> mRNA is correlated with invasive cell behavior in <i>in vitro</i> models; and <i>SYK</i> loss has been correlated with distant metastases in patients. Here, allelic loss of the <i>SYK</i> gene was explored in breast ductal carcinoma in situ (DCIS) using fluorescence <i>in situ</i> hybridization and pyrosequencing, respectively, and in infiltrating ductal carcinoma (IDC) using genomic data from The Cancer Genome Atlas (TCGA). Allelic loss was present in a subset of DCIS cases where adjacent IDC was present. <i>SYK</i> copy number loss was found in about 26% of 1002 total breast cancer cases and 30% of IDC cases. Quantitative immunofluorescence revealed Syk protein to be six-fold higher in infiltrating immune cells compared with epithelial cells. This difference distorted tumor cell mRNA and protein levels in extracts. 20% of 1002 IDC cases contained elevated immune cell infiltration as estimated by elevated immune-specific mRNAs. In cases without immune cell infiltration, loss of <i>SYK</i> copy number was associated with a significant reduction of <i>SYK</i> mRNA. Here we define a 55 Gene Set consisting of Syk interacting, motility- and invasion-related genes. We found that overall survival was significantly reduced in IDC and Luminal A+B cases where copy number and mutations of these 55 genes were affected (Kaplan-Meier, Logrank test p-value 0.007141 and Logrank test p-value 0.001198, respectively). We conclude that reduction in Syk expression and contributions of genomic instability to copy number and mutations in the 55 Syk interacting genes significantly contribute to poorer overall patient survival. A closer examination of the role of Syk interacting motility and invasion genes and their prognostic and/or causative association with metastatic disease and patient outcome is warranted.</p></div

Stratification of cases by breast cancer subtype and by <i>SYK</i> copy number and frequency of alteration.

<p><b>A.</b> The % of altered IDC cases was determined using the 55 Gene Set for cases identified as basal-like, her2, luminal A or luminal B using the PAM50 gene set <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087610#pone.0087610-Cancer1" target="_blank">[28]</a>. <b>B.</b> The average SYK copy number change (CN) was determined for cases identified as in <b>A.</b></p

DNA FISH Analysis.

<p><b>A.</b> Normal control metaphase spreads were used to validate the <i>SYK</i> probe (chromosome 9). The centromeric probe for chromosome 9 is green and the probe for <i>SYK</i> is red. Each BAC was labeled with Spectrum Orange™ (a) RP11-367F26, (b) RP11-83L6, (c) RP11-61N16. <b>B.</b> Confocal microscopy imaging of FISH slides. The panel at left is an example of normal mammary tissue and the panel at right is of DCIS tissue with <i>SYK</i> gene loss. In both, (a) DAPI-stained nuclei, (b) differential interference contrast imaging of section, (c) <i>SYK</i> FISH signal (green), (d) chromosome 9 centromere signal (red). <b>C.</b> The mean value for the ratio of <i>SYK</i>/chromosome 9 centromere signals for each case is illustrated in a bar plot for benign only, DCIS only, and DCIS with adjacent IDC samples. The mean was determined for each case from the ratios obtained individually from 30 cells. Error bars indicate S.E. of the mean. The mean <i>SYK</i>/control ratio of the combined cases was 0.92+/−0.048 S.E. for DCIS only tissues and 0.70+/−0.057 S.E. for DCIS adjacent to IDC cases. The mean for benign only tissues was 0.80+/−0.050 S.E.. The ANOVA P value for the three tissues was 0.035.</p