Serum microRNA expression as an early marker for breast cancer risk in prospectively collected samples from the Sister Study cohort

Ashley C Godfrey, Zongli Xu, Clarice R Weinberg, Robert C Getts, Paul A Wade, Lisa A DeRoo, Dale P Sandler, Jack A Taylor, Ashley C Godfrey, Zongli Xu, Clarice R Weinberg, Robert C Getts, Paul A Wade, Lisa A DeRoo, Dale P Sandler, Jack A Taylor

Abstract

Introduction: MicroRNAs (miRNAs) are small, non-coding, single-stranded RNAs between 18-22 nucleotides long that regulate gene expression. Expression of miRNAs is altered in tumor compared to normal tissue; there is some evidence that these changes may be reflected in the serum of cancer cases compared to healthy individuals. This has yet to be examined in a prospective study where samples are collected before diagnosis.

Methods: We used Affymetrix arrays to examine serum miRNA expression profiles in 410 participants in the Sister Study, a prospective cohort study of 50,884 women. All women in the cohort had never been diagnosed with breast cancer at the time of enrollment. We compared global miRNA expression patterns in 205 women who subsequently developed breast cancer and 205 women who remained breast cancer-free. In addition within the case group we examined the association of miRNA expression in serum with different tumor characteristics, including hormone status (ER, PR, and HER-2) and lymph node status.

Results: Overall, 414 of 1,105 of the human miRNAs on the chip were expressed above background levels in 50 or more women. When the average expression among controls was compared to cases using conditional logistic regression, 21 miRNAs were found to be differentially expressed (P≤.05). Using qRT-PCR on a small, independent sample of 5 cases and 5 controls we verified overexpression of the 3 highest expressing miRNAs among cases, miR-18a, miR-181a, and miR-222; the differences were not statistically significant in this small set. The 21 differentially expressed miRNAs are known to target at least 82 genes; using the gene list for pathway analysis we found enrichment of genes involved in cancer-related processes. In a separate case-case analyses restricted to the 21 miRNAs, we found 7 miRNAs with differential expression for women whose breast tumors differed by HER-2 expression, and 10 miRNAs with differential expression by nodal status.

Conclusions: miRNA levels in serum show a number of small differences between women who later develop cancer versus those who remain cancer-free.

Figures

Figure 1
Figure 1
A large number of microRNAs (miRNAs) are detected in serum. Box-and-whisker plots showing the log (2)-normalized expression for the 47 miRNAs which are expressed above background in 400 individuals. Expression levels were adjusted for batch and chip lot across all samples. The black line represents the median, and the upper and lower 25% are the top and bottom of the box, respectively. Dots represent the outliers.
Figure 2
Figure 2
Serum microRNA (miRNA) expression is associated with tumor subtype. (A) Serum miRNAs significantly associated with HER-2 expression (negative differences correspond to lower levels in women developing tumors with overexpression) (P ≤0.05). (B) Serum miRNAs significantly associated with nodal status (pN1 or higher versus pN0) (P ≤0.05). P values and percentage change were determined by using a linear mixed model.

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