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Supplementary materials for
Circulating miRNA biomarkers: specificity, reproducibility, and the contribution of
extracellular vesicles
Kenneth W. Witwer
Methods: BC miRNA biomarker search
A PubMed search was performed to find articles with the terms microRNA and "breast cancer"
in the title or abstract and at least one of the terms plasma, serum, and blood. 76 publications
were returned. Of these, 8 reviews were excluded, along with several additional reviews that
were not tagged as such in the database. Articles were excluded if examination of the abstract
and/or article did not disclose that one or more miRNAs were measured in plasma or serum.
Articles with whole blood as a substrate were excluded, since almost all miRNAs in whole blood
are found in blood cells and thus are not produced by cancer in tissues. Also, one paper that
consisted of only a small RNA-Seq comparison of one BC and one healthy library (finding well
over 100 putatively different miRNAs) was not considered. Results that included comparisons of
healthy controls and breast cancer patients, or of patients before and after therapy or resection,
were entered in a spreadsheet. Information entered was: miRNA, magnitude and direction of
change (if indicated), fluid substrate (serum, plasma, whole blood), PMID, normalization method
or reference RNA (if indicated and relevant), and comments. In all, the study included 11
publications previously reviewed by Leidner, et al (and examining plasma or serum), the Leidner
data, and 20 studies published since the Leidner, et al cutoff (July, 2012) (1-32).
Supplementary Table I*
miRNA Mean
Citations
FC
-3.4
let-7a
(10,27)
-18.6
19b
(10,27)
-19.0
20a
(10,27)
15.6
25
(12,27)
4.5
27b
(10,27)
-3.5
29a
(9,27)
-14.0
93
(26,27)
-13.2
126
(10,24,27)
-9.0
126*
(10,27)
-14.2
181c
(10,27)
*These miRNAs were concordantly regulated across two or more studies, with no specifically
contradicting results. Note that four of the five studies are from the same university, and three
include including overlapping authors.
Additional miRNAs of note
miR-10b was up in three studies (4- to 200-fold), although in one study, every miRNA examined
was higher in BC serum. miR-10b was slightly down in a fourth study. A fifth determined that
miR-10b was sufficiently stable across healthy and breast cancer samples that it, along with miR30a, was the best normalizer. The evidence for miR-10b is tenuous, despite its apparent role in
tissue and the possibility that it might identify metastasis (27).
miR-16 was found to be upregulated from 3- to 64-fold in three studies, but in many qPCRbased publications, miR-16 was used as the reference miRNA for normalization, sometimes with
apparently rigorous validation. miR-16 might deserve additional scrutiny, if only to sort out its
usefulness in normalization.
miR-21 was reported in 9 studies. In 7, it was upregulated by 1.2 to 200 fold, in one, the
direction of regulation was unclear, and in one, miR-21 was downregulated 4-fold. Although we
labeled miR-21 as "discordant," the weight of the evidence indicates upregulation of extracellular
circulating miR-21 in BC plasma and serum. However, miR-21 is unlikely to be a specific breast
cancer marker. miR-21 was also associated with benign tumors (26) and is one of the most
commonly reported biomarkers for other cancers and a variety of non-neoplastic conditions. It
would be useful in breast cancer only in combination with other, specific markers.
miR-126, an endothelial cell marker, was downregulated 2- to 33-fold in three studies, and its
star arm (miR-126*) was also downregulated. These findings might merit further research into
how the endothelia sense and signal breast cancer states.
miR-145 was slightly downregulated in two studies, but upregulated 2000-fold in another and
found to be unchanged in a fourth.
miR-155, a common marker of inflammation, was upregulated in three studies but
downregulated in two others; others found it unchanged.
miR-223, enriched in myeloid cells, was upregulated very modestly, from 1.2- to 2.4-fold, in
three studies. The small fold changes would be difficult to reproduce.
miR-451 was up- (3) or downregulated (1); miR-451 is almost exclusively expressed in red
blood cells and is an indicator of hemolysis or RBC contamination. As such, it is unlikely to be a
specific breast cancer marker.
Literature included in search
1. Anfossi S, Giordano A, Gao H, Cohen EN, Tin S, Wu Q, et al. High serum miR-19a levels are
associated with inflammatory breast cancer and are predictive of favorable clinical
outcome in patients with metastatic HER2+ inflammatory breast cancer. PLoS One
2014;9:e83113.
2. Appaiah HN, Goswami CP, Mina LA, Badve S, Sledge GW, Jr., Liu Y, et al. Persistent
upregulation of U6:SNORD44 small RNA ratio in the serum of breast cancer patients.
Breast Cancer Res 2011;13:R86.
3. Asaga S, Kuo C, Nguyen T, Terpenning M, Giuliano AE, Hoon DS. Direct serum assay for
microRNA-21 concentrations in early and advanced breast cancer. Clin Chem
2011;57:84-91.
4. Chan M, Liaw CS, Ji SM, Tan HH, Wong CY, Thike AA, et al. Identification of circulating
microRNA signatures for breast cancer detection. Clin Cancer Res 2013;19:4477-87.
5. Chen W, Cai F, Zhang B, Barekati Z, Zhong XY. The level of circulating miRNA-10b and
miRNA-373 in detecting lymph node metastasis of breast cancer: potential biomarkers.
Tumour Biol 2013;34:455-62.
6. Cuk K, Zucknick M, Madhavan D, Schott S, Golatta M, Heil J, et al. Plasma microRNA panel
for minimally invasive detection of breast cancer. PLoS One 2013;8:e76729.
7. Freres P, Josse C, Bovy N, Boukerroucha M, Struman I, Bours V, et al. Neoadjuvant
chemotherapy in breast cancer patients induces miR-34a and miR-122 expression. J Cell
Physiol 2014.
8. Gezer U, Keskin S, Igci A, Tukenmez M, Tiryakioglu D, Cetinkaya M, et al. Abundant
circulating microRNAs in breast cancer patients fluctuate considerably during
neoadjuvant chemotherapy. Oncol Lett 2014;8:845-8.
9. Godfrey AC, Xu Z, Weinberg CR, Getts RC, Wade PA, DeRoo LA, et al. Serum microRNA
expression as an early marker for breast cancer risk in prospectively collected samples
from the Sister Study cohort. Breast Cancer Res 2013;15:R42.
10. Guo L, Zhao Y, Yang S, Cai M, Wu Q, Chen F. Genome-wide screen for aberrantly
expressed miRNAs reveals miRNA profile signature in breast cancer. Mol Biol Rep
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11. Guo LJ, Zhang QY. Decreased serum miR-181a is a potential new tool for breast cancer
screening. Int J Mol Med 2012;30:680-6.
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cancer risk: the use of miR-484/191 as endogenous controls. Carcinogenesis
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13. Kodahl AR, Lyng MB, Binder H, Cold S, Gravgaard K, Knoop AS, et al. Novel circulating
microRNA signature as a potential non-invasive multi-marker test in ER-positive earlystage breast cancer: a case control study. Mol Oncol 2014;8:874-83.
14. Kodahl AR, Zeuthen P, Binder H, Knoop AS, Ditzel HJ. Alterations in circulating miRNA
levels following early-stage estrogen receptor-positive breast cancer resection in postmenopausal women. PLoS One 2014;9:e101950.
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16. Leidner RS, Li L, Thompson CL. Dampening enthusiasm for circulating microRNA in breast
cancer. PLoS One 2013;8:e57841.
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biomarkers for breast cancer detection. PLoS One 2013;8:e53141.
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