Circulating biomarkers can provide a less invasive way to monitor patients and their response to therapies. Recent advances in next generation RNA sequencing have allowed for a comprehensive characterization of the transcribed genome, which extends past protein-coding genes to include non-coding RNAs, important regulators of protein-coding gene expression. While several studies have shown that circulating small non-coding RNAs, specifically microRNAs, have the potential to serve as biomarkers of cancer, little work has been done to study the role of long RNAs as circulating biomarkers. Our goal was to identify the role of mRNAs and long non-coding RNAs as biomarkers of detection and response to therapy for breast cancer.
To start, we utilized samples from 13 breast cancer patients whose tumors were biopsied for research prior to and after trastuzumab treatment as part of a clinical trial. RNA was extracted from both the pre- and post-treatment biopsies for each patient. Next generation RNA-sequencing was completed and reads aligned to known mRNAs and lncRNAs. Mean expression levels were inferred and the statistical significance of differences in expression between pre- and post-treatment samples was assessed among the 11 women in the trial that achieved a pathological complete response. We then compared this to the circulating transcriptome from 10 breast cancer patients and 10 age and race matched mammography-screened controls, which provided blood samples for biomarker studies as part of a larger case-control study. RNA was extracted from the plasma on all 20 samples. Next generation RNA-sequencing was done and was aligned to known mRNAs and lncRNAs as in the tumor samples from the clinical trial. Differences in circulating RNA expression between the breast cancer patients and controls was done using a t-test.
After exclusion of RNAs that were expressed in <25% of the samples, the expression level of 163 lncRNAs and 1702 mRNAs were associated with treatment (p<0.05). Of the mRNAs associated with treatment response, 9 that were expressed at higher levels before treatment were also increased in the circulation in breast cancer patients, compared to controls (p<0.05). None of the lncRNAs that were altered in response to treatment were statistically significantly differentially expressed in the circulation, although we are limited in our ability to rule this out due to sample size. Validation of these mRNAs in plasma in a larger sample of cases and controls, as well as in patients before and after treatment, is currently ongoing.
Our data shows that, as expected, the expression profile of both mRNAs and lncRNAs in breast cancer tissue changes with treatment, and transcriptome profiling of RNA from the plasma suggests that many of these same RNAs are deregulated in the circulation of breast cancer patients. More work will need to be done to see if the circulating transcriptome can be used as a marker for early breast cancer diagnosis or response to treatment.
Citation Format: Cheryl L. Thompson, Ahmad M. Khalil, Kristy Miskimen, Callie Merry, Lyndsay Harris. The tumor and circulating transcriptomes in breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3487. doi:10.1158/1538-7445.AM2013-3487