Conceived and designed the experiments: MGS RS RS-W A. Hartmann A. Hein MWB PAF. Performed the experiments: MGS RS PLS LK SR. Analyzed the data: MGS RS LK CRL CMB. Contributed reagents/materials/analysis tools: MPL SMJ MRB SR BRA EW CR. Wrote the paper: MGS RS MWB PAF.
The authors have declared that no competing interests exist.
MicroRNAs (miRNAs, miRs) are a class of small, non-coding RNA molecules with relevance as regulators of gene expression thereby affecting crucial processes in cancer development. MiRNAs offer great potential as biomarkers for cancer detection due to their remarkable stability in blood and their characteristic expression in many different diseases. We investigated whether microarray-based miRNA profiling on whole blood could discriminate between early stage breast cancer patients and healthy controls.
We performed microarray-based miRNA profiling on whole blood of 48 early stage breast cancer patients at diagnosis along with 57 healthy individuals as controls. This was followed by a real-time semi-quantitative Polymerase Chain Reaction (RT-qPCR) validation in a separate cohort of 24 early stage breast cancer patients from a breast cancer screening unit and 24 age matched controls using two differentially expressed miRNAs (miR-202, miR-718).
Using the significance level of p<0.05, we found that 59 miRNAs were differentially expressed in whole blood of early stage breast cancer patients compared to healthy controls. 13 significantly up-regulated miRNAs and 46 significantly down-regulated miRNAs in our microarray panel of 1100 miRNAs and miRNA star sequences could be detected. A set of 240 miRNAs that was evaluated by radial basis function kernel support vector machines and 10-fold cross validation yielded a specificity of 78.8%, and a sensitivity of 92.5%, as well as an accuracy of 85.6%. Two miRNAs were validated by RT-qPCR in an independent cohort. The relative fold changes of the RT-qPCR validation were in line with the microarray data for both miRNAs, and statistically significant differences in miRNA-expression were found for miR-202.
MiRNA profiling in whole blood has potential as a novel method for early stage breast cancer detection, but there are still challenges that need to be addressed to establish these new biomarkers in clinical use.
Breast cancer (BC) is one of the leading causes of cancer death among women worldwide
MicroRNAs (miRNAs, miRs) are a novel class of endogenous, non-coding, single-stranded RNAs, first described in 1993 by Lee et al. in C. elegans
MiRNA loci are statistically over-represented at fragile genomic regions that are commonly amplified or deleted in human cancers, implying a connection of miRNAs with cancer initiation and progression
The finding of a decrease of miR-15a and miR-16-1 in patients with chronic lymphocytic leukaemia was one of the first direct links between regulative miRNAs and cancer
Aim of the present study was to analyze the miRNA expression patterns in whole blood of patients with early stage BC in comparison to healthy controls using a miRNA microarray chip.
All patients of this study participated in a prospective case control study for the molecular detection of breast cancer (MODE-B Study). Patients presenting at our specialized breast cancer unit with suspect breast lesions are routinely asked to participate in this still ongoing MODE-B Study. The presented data are results from the monocentric miRNA pilot-study.
The MODE-B Study was approved by the Ethics Committee of the Medical Faculty of the Friedrich-Alexander University Erlangen-Nuremberg (reference number 3937). Written informed consent was obtained from every patient and control individual before blood was taken.
Venous blood samples (non-fasting) (2.7 mL per patient) were collected from cases and controls in EDTA blood tubes (Sarstedt, Monovette EDTA K; Sarstedt AG, Germany) containing 1.6 mg EDTA as anticoagulant and stored at −20°C until further processing. We selected the first 48 consecutive early stage BC patients suitable for this prospectively planed miRNA-biomarker-study.
This study comprises two case control studies, a discovery study (microarray chip analysis) and a validation study (RT-qPCR of selected miRNAs). Patients were included in the specialized breast unit. They either referred themselves because of a newly palpable breast lesion or were referred to the breast unit by their physicians because of suspicious lesions for further breast diagnostics and biopsy.
The independent RT-qPCR validation cohort consisted of consecutive early stage BC patients diagnosed within the German BC screening program. These differences in patients' recruitment between the microarray study cohort and the RT-qPCR validation cohort resulted inevitably in a lower risk profile of patients in the validation cohort (
Parameters | Microarray cohort patients (%) | RT-qPCR validation cohort patients (%) |
Total | 48 | 24 |
Age | 61.9 (range 34–89 years) | age matched pairs |
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pT1a | 0 | 3 (12.5) |
pT1b | 12 (25.0) | 6 (25.0) |
pT1c | 29 (60.4) | 11 (46.0) |
pT2 | 7 (14.6) | 4 (16.5) |
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pN0 | 39 (81.25) | 23 (96.0) |
pN1 (1–3 lymph nodes) | 9 (18.75) | 1 (4.0) |
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G1 | 12 (25.0) | 10 (42.0) |
G2 | 19 (39.6) | 12 (50.0) |
G3 | 17 (35.4) | 2 (8.0) |
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Positive | 44 (91.7) | 21 (87.5) |
Negative | 4 (8.3) | 3 (12.5) |
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Positive | 39 (81.25) | 16 (67.0) |
Negative | 9 (18.75) | 8 (33.0) |
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Positive | 40 (83.3) | 1 (4.0) |
Negative | 8 (16.7) | 23 (96.0) |
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11 (22.9) | 4 (16.7) |
10 | 13 (27.1) | 11 (45.8) |
10–20 | 10 (20.9) | 6 (25.0) |
>20 | 14 (29.1) | 3 (12.5) |
Total RNA extraction was performed as published previously
The quality and quantity of the RNA was evaluated by 260/280 ratio using NanoDrop spectrophotometry (NanoDrop ND-1000) and Agilent 2100 Bioanalyzer (Agilent Technologies Inc., Santa Clara, CA).
Each array of the Geniom® Biochip contains 11 replicates of 1100 miRNAs and miRNA star sequences as annotated in the Sanger database miRBase15.0
The approximate normal distribution of the measured data was verified by Shapiro–Wilk test with a median P-value of 5.9E-20. MiRNAs with different expression levels between BC patients and controls were identified by unpaired two-tailed parametric t-test. P-values obtained for each individual miRNA were adjusted for multiple testing by Benjamini–Hochberg adjustment
miRNAs | Median controls | Median BC pat. | Fold change | T-test raw P-value | T-test adj P-value | AUC |
miR-4306 | 10.09 | 10.83 | 2.08 | 0.0007 | 0.02 | 0.71 |
miR-202 | 4.49 | 5.21 | 2.04 | 0.0004 | 0.02 | 0.72 |
miR-4257 | 5.50 | 6.17 | 1.96 | 0.0017 | 0.04 | 0.65 |
miR-1323 | 5.48 | 6.13 | 1.92 | 0.0022 | 0.04 | 0.69 |
miR-335 | 7.52 | 8.16 | 1.89 | 0.0017 | 0.04 | 0.74 |
miR-497 | 7.28 | 7.87 | 1.82 | 6.56E-05 | 0.01 | 0.75 |
miR-106b | 13.22 | 13.76 | 1.69 | 0.0004 | 0.01 | 0.72 |
miR-922 | 6.98 | 7.49 | 1.67 | 0.0019 | 0.03 | 0.65 |
miR-516b | 5.59 | 6.09 | 1.64 | 0.0008 | 0.03 | 0.67 |
let7a* | 5.10 | 5.40 | 1.35 | 0.001 | 0.03 | 0.65 |
BC pat. … breast cancer patients; adj … adjusted.
miRNAs | Median controls | Median BC pat. | Fold change | T-test raw P-value | T-test adj P-value | AUC |
miR-718 | 7.26 | 6.12 | 3.12 | 4.27E-05 | 0.0041 | 0.77 |
miR-625* | 7.57 | 6.46 | 3.05 | 1.19E-05 | 0.0020 | 0.77 |
miR-1471 | 6.41 | 5.61 | 2.24 | 0.0002 | 0.0127 | 0.70 |
miR-193a-3p | 7.34 | 6.57 | 2.16 | 1.67E-07 | 0.0001 | 0.79 |
miR-182 | 12.67 | 11.91 | 2.14 | 0.0001 | 0.0080 | 0.71 |
miR-1915 | 8.73 | 8.05 | 1.98 | 1.53E-05 | 0.0022 | 0.75 |
miR-564 | 7.26 | 6.60 | 1.93 | 0.0003 | 0.0127 | 0.67 |
miR-107 | 12.41 | 11.80 | 1.83 | 0.00193 | 0.0414 | 0.68 |
miR-2355 | 7.15 | 6.55 | 1.82 | 4.91E-05 | 0.0042 | 0.73 |
miR-3186-3p | 6.96 | 6.37 | 1.81 | 6.39E-06 | 0.0015 | 0.75 |
miR-24 | 11.54 | 10.94 | 1.81 | 0.0007 | 0.0235 | 0.65 |
miR-3130-3p | 7.89 | 7.29 | 1.81 | 5.03E-05 | 0.0042 | 0.73 |
miR-526a | 7.33 | 6.77 | 1.76 | 0.0003 | 0.0127 | 0.72 |
miR-1469 | 7.17 | 6.64 | 1.70 | 0.0001 | 0.0080 | 0.68 |
miR-874 | 7.53 | 7.01 | 1.67 | 2.10E-06 | 0.0006 | 0.74 |
BC pat. … breast cancer patients; adj … adjusted.
Two miRNA (miR-202, miR-718) which were differentially expressed in the microarray assays were analyzed in an independent validation cohort by RT-qPCR. Among the most deregulated miRNAs from our microarray experiments we selected miRNAs for PCR-validation with purchasable and well established primers to reduce sources of error. The miRNA isolation and RNA quantification of the independent validation cohort was identical to the microarray analysis. TaqMan miRNA assays from ABI (Applied Biosystems, Foster City, CA, USA) were purchased for miR-202 and miR-718. First, each miRNA was specifically reverse transcribed according to manufactures protocol using TaqMan miRNA RT-Kit with stem-loop RT-primer and ABI7300 (ABI). Second, each sample was analyzed in duplicates for each specific miRNA using a RT-primer with universal master mix II (without Uracil-N-glycosylase) on the Applied Biosystems 7300 Sequence Detection System according to manufactures protocol. The cycle thresholds (Ct) for BC patients and their age-matched healthy controls were calculated and normalized to miR-16 (miR-16; ABI), which was found in the literature as the most widely-used endogenous control miRNA for RT-qPCR. Each analysis also contained inter- and intra-assay replicates. High Ct-values indicated low miRNA quantity and vice versa. The expression levels of miRNAs in BC patients relative to their age-matched healthy controls were calculated using the comparative cycle threshold (
In total, 153 whole blood samples of early stage BC patients and healthy control individuals were analyzed in this study. Clinical patient and tumor characteristics at time of BC diagnosis are shown in
In the discovery setting, miRNA-microarray analyses of 48 BC cases using the Geniom® Realtime Analyzer microarray platform identified 59 deregulated miRNAs in whole blood of early stage BC patients compared to healthy controls
The graph shows the logarithm of the quotient of the probability to be BC sample and the probability to be a control sample (log odds) of all samples analyzed with miRNA-microarrays. 1… Controls; 2…BC patient.
The expression levels of two miRNAs with purchasable and well established primers were confirmed with a Taqman-based RT-qPCR in an independent cohort of BC patients and controls using individual miRNA-specific primers. MiR-202 which showed up-regulation pattern for BC cases over controls in the microarray analyzes and miR-718 which was identified with down-regulation pattern were selected to be validated in an independent cohort of 24 age matched pairs of early stage BC patients and 24 healthy controls. We were able to validate the microarray separation pattern of the two miRNAs in the validation cohort using RT-qPCR. The PCR results are summarized in
miRNA | Relative fold change RT-qPCR | T-test P-value RT-qPCR validation | Changes in BC cases | Fold change microarray |
miR-202 | 19.38 | 0.03 | Up-regulation | 2.04 |
miR-718 | 5.44 | 0.72 | Down-regulation | 3.12 |
RT-qPCR results were concordant with the miRNA microarray results in terms of up- and down-regulation calculated as relative fold changes in comparison with each age matched control and calculated as relative fold changes between the two groups (
Significant different expression of circulating miR-202 in whole blood of BC patients versus Controls (up-regulation of miR-202). Data derived from RT-qPCR and presented as delta-Ct values, with higher values standing for lower miRNA-expression.
Mammography is currently the modality of choice for screening for early BC and possesses a sufficient sensitivity and specificity. Specificity of screening mammography is over 95%, but sensitivity ranges between 67% and 95% and is strongly dependent on several factors like age, breast density and professional experience of the examiner
Several miRNAs are known to be aberrantly expressed in human BC tissue and have been correlated with clinical stage and clinico-pathological variables like hormone receptor status, tumor subtypes, as well as clinical variables like metastatic potential, progression free survival and overall survival
Blood-based miRNA-profiling is still far behind the improvements in tissue-based miRNA-profiling, but offers the potential for early, non-invasive, sensitive and specific BC detection and screening. First reports using serum or plasma for RT-PCR or microarray based miRNA-profiling were promising. Recently, a serum based study using next-generation sequencing of miRNAs for BC detection has also been reported and several miRNAs have been identified as potential serum/plasma biomarkers in different cancer types like lung, prostate, colon and liver cancer
One potential advantage of the whole blood approach could be the higher miRNA-content and the chance to measure not only tumor secreted oncogenic miRNAs, but also the changes in the miRNA profile following the “host-reaction” in the body of the patient
The comparison of miRNA-profiles from whole blood and plasma/serum is also addressed in the work of Zhao et al.
The median fold expression changes of deregulated miRNAs in our microarray analyses were in the range of 2 to 4 fold which is in line with previous miRNA microarray profiling studies, but fare less than previously reported in qRT-PCR based studies. These differences were expected and are probably due to the different detection and analysis techniques. The relative fold changes found in our RT-qPCR validation analyses were higher, with miR-202 and miR-718 showing a relative fold change of about 20 and over 5 between cases and age-matched controls (
The clinicopathological characteristics of the two cohorts are slightly different with smaller and less aggressive tumors with a higher rate of HER2-negativity in the RT-qPCR cohort compared to the microarray cohort. This is due to the different recruitment strategy (hospital based versus screening cohort) and the consecutive patient recruitment.
We used miRNA microarray technology to analyze the miRNA expression profiles of early stage BC patients compared to healthy controls from frozen EDTA-whole-blood and validated the results with RT-qPCR in an independent early stage BC cohort. Research of circulating miRNAs as blood based biomarkers is still in its infancy. However, this study as well as other recent studies indicate that miRNA-analyses have diagnostic and prognostic potential and could improve early stage BC detection in the future. There are several possible applications of miRNA profiling conceivable in the future. Firstly, miRNA profiles could help to reduce unnecessary breast biopsies if miRNA sets could be identified which reliably identify BC free individuals. Secondly, miRNA profiling could be used as a pre-screening method for example by general practitioners to identify women with an urgent need for breast diagnostics. Thirdly, in younger patients with dense breast tissue a future miRNA-based BC screening could possibly provide better sensitivity and specificity than the mammography even without radiation exposure.
Our study detected several significant deregulated miRNAs in frozen whole blood of early stage BC patients, which should be analyzed further with regard to their function in breast cancer development and progression. Moreover, large prospective clinical studies are clearly warranted to confirm our preliminary results and further explore the existing potential of circulating miRNAs in serum, plasma or whole blood as diagnostic and therapeutic BC biomarkers.
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We thank Ms. Elisabeth Stiegler, Sonja Oeser, Silke Landrith and the febit biomed team for their expert technical assistance.