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Address correspondence to Lorenzo F. Sempere, Ph.D., Laboratory of microRNA Diagnostics and Therapeutics, Van Andel Research Institute, 333 Bostwick Ave NE, Grand Rapids, MI 49503.
Center for Cancer and Cell Biology, Van Andel Research Institute, Grand Rapids, MichiganLaboratory of microRNA Diagnostics and Therapeutics, Van Andel Research Institute, Grand Rapids, Michigan
Triple-negative breast cancer (TNBC) is an aggressive subtype defined by the lack of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 expression. Expression of miR-21, an oncomiR, is frequently altered and may be distinctly expressed in the tumor stroma. Because tumor lesions are a complex mixture of cell types, we hypothesized that analysis of miR-21 expression at single-cell resolution could provide more accurate information to assess disease recurrence risk and BC-related death. We implemented a fully automated, tissue slide–based assay to detect miR-21 expression in 988 patients with BC. The miR-21High group exhibited shorter recurrence-free survival [hazard ratio (HR), 1.71; P < 0.001] and BC-specific survival (HR, 1.96; P < 0.001) in multivariate regression analyses. When tumor compartment and levels of miR-21 expression were considered, significant associations with poor clinical outcome were detected exclusively in tumor epithelia from estrogen receptor– and/or progesterone receptor–positive human epidermal growth factor receptor 2–negative cases [recurrence-free survival: HR, 3.67 (P = 0.006); BC-specific survival: HR, 5.13 (P = 0.002)] and in tumor stroma from TNBC cases [recurrence-free survival: HR, 2.59 (P = 0.013); BC-specific survival: HR, 3.37 (P = 0.003)]. These findings suggest that the context of altered miR-21 expression provides clinically relevant information. Importantly, miR-21 expression was predominantly up-regulated and potentially prognostic in the tumor stroma of TNBC.
Breast cancer (BC) is a highly heterogeneous disease consisting of several molecular subtypes that correlate with clinical outcome. Expression levels of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) are used to define the major subtypes, with ER+ tumors having a significantly better prognosis than ER− tumors.
Patients with BC have benefited from recent advances in molecular diagnostic assays. For ER+ cases, assays that are gene expression based (ie, OncoType Dx, Mammaprint, and PAM50) and tissue slide based [eg, immunohistochemistry (IHC) 4 and Mammostrat] can refine risk assessment for recurrence and identify patients who may benefit from intensive adjuvant treatments in addition to hormonal therapy.
Mammostrat as an immunohistochemical multigene assay for prediction of early relapse risk in the tamoxifen versus exemestane adjuvant multicenter trial pathology study.
Subtyping of breast cancer by immunohistochemistry to investigate a relationship between subtype and short and long term survival: a collaborative analysis of data for 10,159 cases from 12 studies.
This is particularly relevant for triple-negative BC (TNBC), which accounts for 15% to 20% of BC cases and is defined by the lack of ER, PR, and HER2 expression.
TNBCs are associated with shorter recurrence free survival (RFS) and BC-specific survival (CSS) relative to other major BC subtypes. TNBC is still a heterogeneous group of diseases, and gene expression profiling methods have suggested the existence of distinct subgroups.
Currently, TNBC is treated with different cytotoxic combination chemotherapy; complete response to neoadjuvant chemotherapy, depending on specific treatment regimens, varies widely, between 8% and 83%.
Thus, predictive biomarkers for therapeutic response prediction and novel therapeutic targets that address distinct biological features of TNBC subgroups are needed for these patients.
Interactions between tumor epithelia (cancer cells) and the tumor microenvironment (TME) play a major role in BC progression. The TME is composed of a complex network of stromal cells, immune cells, extracellular matrix, and cytokines/chemokines. Stromal gene expression signatures have been identified that predict outcome and treatment response in BC.
Despite the overwhelming data on the influence of the TME, cancer diagnostics and therapeutics are directed primarily at targeting the tumor epithelia.
miRNAs are a class of short, noncoding, regulatory RNAs that modulate gene expression in important developmental, physiological, and pathological processes.
A single miRNA can down-regulate the expression of hundreds of target genes by binding to a partially complementary site in the 3′-untranslated region of their cognate mRNAs. The clinical implications of miRNA biological features are generating great interest in the areas of cancer research and cancer medicine.
Sempere LF, Kauppinen S: Translational Implications of MicroRNAs in Clinical Diagnostics and Therapeutics: Handbook of Cell Signaling. Edited by RA Bradshaw, EA Dennis. Oxford, Academic Press, 2009, pp 2965–2981
miRNA-based diagnostics is an emerging area of novel prognostic and predictive indicators, and some miRNAs have provided leads for the development of novel targeted therapies.
Quantitative real-time RT-PCR expression analysis of miRNAs in formalin-fixed tissue specimens are now offered commercially as ancillary tests for identifying the organ site of cancers of unknown primary, for performing differential diagnosis of pancreatic cancer, and for obtaining subtype classification of lung and renal cell cancers.
Both in vitro and in vivo work has demonstrated the significant role that miR-21 plays in tumorigenesis and the potential of miR-21 as a therapeutic target.
miR-21 also exerts fibrogenic functions, promoting fibrosis in mouse models of renal and cardiac injury–induced failure as well as in in vitro co-culture studies of colon and pancreatic cancer cell lines.
It is not clear whether miR-21 exerts its tumorigenic functions in BC primarily within cancer cells or in other cellular elements of the TME. Research using in situ hybridization (ISH) has suggested that miR-21 expression is predominantly up-regulated in the tumor stroma.
The few BC cases in these studies precluded prognostic evaluation of this marker.
Herein, we conducted an adequately powered ISH-based study on 901 BC cases to test the hypothesis that both the levels and tumor compartment of miR-21 expression in primary BC tumor tissues are informative for predicting RFS and CSS. We observed that miR-21 expression was significantly associated with clinical outcome in different BC subtypes on the basis of ER-PR/HER2 status. This association was tumor compartment specific and was influenced by the intrinsic biological features of major BC subtypes. Specifically, high expression of miR-21 in the tumor epithelia in ER+ and/or PR+HER2− cases was associated with poor clinical outcome, and high expression of miR-21 in tumor stroma was associated with a much poorer clinical outcome in TNBC.
Materials and Methods
Patient Cohort
Tissue microarrays (TMAs) representing tissue cores from 988 female patients diagnosed with nonmetastatic invasive BC between 1985 and 1997 (stage I, sets 9 to 13; and stage II, sets 14 to 17) were acquired from the National Cancer Institute (NCI) Cancer Diagnosis Program (CDP; http://cdp.nci.nih.gov/breast/prognostic_cs.html, last accessed September 19, 2014). This NCI CDP 2008 Series Prognostic TMAs include 590 stage I and 398 stage II cases. The Institutional Review Board (Dartmouth College Committee for the Protection of Human Subjects) approved a waiver for the entire consent process and use of a consent form (Committee for Protection of Human Subjects number 22042).
ISH and IHC
A fluorescence-based ISH/IHC assay was conducted as previously described,
with slight modifications, on a Leica BOND-MAX automated staining station (Leica Biosystems, Buffalo Grove, IL) in the Dartmouth Pathology Translational Research Laboratory, a College of American Pathologists’–accredited Clinical Laboratory Improvement Amendments–certified facility. miR-21 staining was calibrated by adjusting probe concentration and fluorescent substrate incorporation time so that no signal was detectable in adjacent normal tissue of BC tumor lesions. Briefly, double-tagged miR-21 (FAM2X) and snRNA U6 (biotin2X) locked nucleic acid–modified DNA probes at 50 nmol/L each were hybridized to tissue slides for 75 minutes at 45°C. Expression of miR-21, U6, and cytokeratin (CK) 19 was assessed with appropriate antibody combinations, followed by sequential rounds of HRP-mediated deposition of appropriate fluorochrome-conjugated tyramine substrates for 20 minutes. Fluorescent images were captured with an EXi Aqua QImaging camera (QImaging, Surrey, BC, Canada) mounted on a BX51 microscope (Olympus, Center Valley, PA). Image-Pro Plus software version 7.0 (Media Cybernetics, Rockville, MD) was used for histogram-based image segmentation analysis. Eighty-seven cores were excluded because of insufficient representation of tumor lesions and/or lack of stain for all markers. miR-21 signal intensity was scored in two locations, tumor epithelium and stroma, on a scale from 0 (no expression) to 3 (high expression). For assessing correlations with clinicopathological factors, sample tissue cores were considered low (0 or 1) or high (2 or 3) for each location (Supplemental Table S1). This scoring system was independently validated by multiviewer (L.F.S. and W.A.W.) and computer-assisted image segmentation analyses on digital images captured from a representative subset of 399 cases (Supplemental Figure S1).
Statistical Analysis
We followed the REMARK guidelines for reporting of prognostic markers
and the NCI CDP's recommendations for data set analysis (http://cdp.nci.nih.gov/breast/prognostic_dm.html, last accessed September 19, 2014). These TMAs have a robust statistical design to detect associations between tumor markers and clinical outcome in nonmetastatic BC cases. TMA sample sizes were determined by NCI statisticians to detect a hazard ratio (HR) of 2.0 with 80% or greater power. Associations between miR-21 expression and clinicopathological factors were assessed by t-test, χ2 test, or Fischer's exact test (Supplemental Appendix S1). Kaplan-Meier curves (which estimate survival if death from other causes were eliminated as a possibility) of RFS and CSS were constructed. RFS was defined as the time to any documented recurrence event or time of death with evidence of BC in the absence of an earlier documented recurrence event; CSS was defined as time before death with evidence of BC. Kaplan-Meier curves were compared using the log-rank test. Cause-specific HRs, subdistribution HRs, and their 95% CIs were derived using the Cox proportional hazards model. Unless otherwise noted, reported HRs correspond to results from the multivariate Cox proportional hazards regression model, adjusted for age, tumor size, lymph node involvement, and grade. All statistical analyses were performed using the R software package (http://www.r-project.org).
Results
miR-21 Is Predominantly Expressed in the Tumor Stroma in Breast Cancer
To investigate the prognostic value of tumor compartment and levels of miR-21 expression in BC, we determined the expression of miR-21, snRNA U6, and CK19 protein on tissue cores represented in the NCI CDP Prognotic TMAs, using a fully automated fluorescence-based multiplex ISH/IHC assay.
Expression of snRNA U6 was used to assess RNA quality, and expression of CK19 was used to identify the tumor epithelia in each tissue core. In total, 901 cases were included in the analysis. Molecular and clinicopathological characteristics are summarized in Table 1, and the complete data set with clinical outcomes is provided in Supplemental Table S1.
Table 1Patient Characteristics and miR-21 Expression Data
Characteristic
Condition
n
% of Total
% of Available
miR-21 compartment
Tumor epithelia expression
45
5.00
5.00
Tumor stroma expression
341
37.80
37.80
No expression
515
57.20
57.20
miR-21 score
High
207
23.00
23.00
Low
694
77.00
77.00
Age (years)
<55
331
36.70
36.70
>55
570
63.30
63.30
Tumor size (cm)
<2
646
71.70
71.70
>2
255
28.30
28.30
LN status
N0
642
71.30
71.30
N1
259
28.70
28.70
Grade
1
249
27.60
27.60
2
412
45.70
45.70
3
240
26.60
26.60
Stage
I
520
57.60
57.60
II
381
42.40
42.40
ER status
Negative
182
20.20
23.82
Positive
582
64.60
76.18
Data not available
137
15.20
NA
PR status
Negative
233
25.90
30.46
Positive
532
59.00
69.54
Data not available
136
15.10
NA
HER status
Negative
658
73.00
85.34
Positive
113
12.50
14.66
Data not available
130
14.40
NA
ER/PR/HER2 subtype
ER+ and/or PR+HER2–
549
60.93
72.14
(any ER or PR) HER2+
107
11.88
14.06
ER–PR–HER2–
105
11.65
13.80
Data not available
140
15.54
NA
Data are given as number and percentage of cases in each category for miR-21 expression data and patient characteristics currently used in the clinic as prognostic factors. Additional patient characteristics are provided in Supplemental Table S1.
ER, estrogen receptor; HER2, human epidermal growth factor receptor 2; LN, lymph node; NA, not applicable because data are missing for these categories; PR, progesterone receptor.
miR-21 expression was detected in 386 (42.8%) of the 901 cases. miR-21 was predominantly or exclusively expressed within the tumor stroma compartment (CK19– cells) in most cases (88.3%), whereas it was confined to the tumor epithelia compartment (CK19+ cells) in a few cases (11.7%). We previously determined by microarray and Northern blot analyses that miR-21 was expressed at high levels in the MCF7 BC cell line and was not expressed in the MCF10A BC line.
Therefore, we used cores of these BC cell lines represented in TMA slides as a reference for scoring miR-21 expression levels (Figure 1). Cases were reviewed under fluorescent microscopy and assigned a score of low (694 cases) or high (207 cases) on the basis of signal intensity. This miR-21 score provided a threshold that further separated nonexpressing and low-expressing cases from those with higher miR-21 expression.
Figure 1Patterns, quantification, and scoring of miR-21 expression in breast cancer lesions. A and B: Representative images of multicolor detection of miR-21, snRNA U6, and CK19 in BC cell lines (A) and tissue cores (B). miR-21 heat map images were generated using histogram-based image segmentation analysis; the background signal was set on the basis of exposed slide areas (eg, tissue holes). Other categories were defined relative to the highest background intensity (black) as onefold (blue), twofold (cyan), threefold (orange), and fourfold or more (red) over background. Cases with a score of 1 to 2 and 3 to 4 were categorized low and high miR-21 expression, respectively. C: Summary of miR-21 expression levels, tumor compartment of expression, and molecular subtype on the basis of ER/PR/HER2 status in entire patient cohort. Original magnification, ×200 (A and B). Ca, tumor epithelia; Stroma, tumor stroma.
miR-21 Score Is an Independent Risk Factor Associated with Poor Disease Outcome
The miR-21High group had an increased risk for shorter RFS (Supplemental Tables S2 and S3) and CSS (Tables 2 and 3) at 5 years after diagnosis and during the overall follow-up period (median, 10.33 years). Stratification of cases by standard clinicopathological characteristics indicated that the miR-21 score was an independent risk factor associated with poor disease outcome (Tables 2 and 3 and Supplemental Tables S4 and S5). Notably, the miR-21High grade 2 subgroup had a much higher risk relative to the miR-21Low grade 2 subgroup for shorter RFS (5 years: HR, 3.85; P < 0.001) and CSS (5 years; HR, 5.51; P < 0.001) and a similar risk as the miR-21Low grade 3 subgroup (Supplemental Table S4).
Table 2CSS Risk Associated with miR-21 Expression for Disease Outcome 5 Years after Diagnosis
Group
Variable
Univariate
Multivariate
HR
95% CI
P value
HR
95% CI
P value
All cases (n = 901)
miR-21High/Low
2.69
1.65–4.39
<0.001
2.31
1.41–3.79
<0.001
miR-21HiCancer/Low
3.86
1.35–11.03
0.010
3.04
1.05–8.86
0.037
miR-21HiStroma/Low
2.57
1.54–4.28
<0.001
2.22
1.32–3.73
0.002
Grade 1/2
0.40
0.132–1.235
0.105
0.48
0.16–1.48
0.195
Grade 3/2
5.53
3.10–9.85
<0.001
4.51
2.50–8.13
<0.001
Stage II/I
4.42
2.52–7.74
<0.001
1.04
0.36–3.05
0.937
ER+ and/or PR+HER2− (n = 549)
miR-21High/Low
3.15
1.46–6.81
0.003
2.57
1.18–5.61
0.016
miR-21HiCancer/Low
9.46
2.65–33.80
<0.001
7.05
1.75–28.44
0.005
miR-21HiStroma/Low
2.62
1.15–6.01
0.020
2.15
0.93–4.99
0.069
ER−PR− HER2− (n = 105)
miR-21High/Low
2.50
1.03–6.08
0.039
2.82
1.10–7.26
0.028
miR-21HiCancer/Low
NA
NA
NA
NA
NA
NA
miR-21HiStroma/Low
2.84
1.17–6.90
0.019
3.09
1.20–7.98
0.017
Data are given as HRs and their 95% CIs for miR-21 expression and standard clinical indicators using Cox proportional hazard models. The following variables were adjusted for in multivariate analyses: age, tumor size, grade, and lymph node involvement. Risks associated with miR-21High score are reported as a whole group (miR-21High) and as subgroups (miR-21HiCancer and miR-21HiStroma) divided by tumor compartment of expression.
NA, not applicable because not enough data entries for this category to perform analysis.
Table 3CSS Risk Associated with miR-21 Expression for Overall Disease Outcome
Group
Variable
Univariate
Multivariate
HR
95% CI
P value
HR
95% CI
P value
All cases (n = 901)
miR-21High/Low
1.94
1.38–2.75
<0.001
1.96
1.38–2.78
<0.001
miR-21HiCancer/Low
2.61
1.13–6.05
0.023
2.74
1.17–6.39
0.018
miR-21HiStroma/Low
1.88
1.31–2.70
<0.001
1.88
1.31–2.72
<0.001
Grade 1/2
0.49
0.29–0.84
0.007
0.56
0.33–0.96
0.032
Grade 3/2
2.57
1.81–3.66
<0.001
2.16
1.51–3.10
<0.001
Stage II/I
3.15
2.23–4.43
<0.001
0.97
0.48–1.94
0.925
ER+ and/or PR+HER2− (n = 549)
miR-21High/Low
1.70
1.05–2.75
0.028
1.81
1.11–2.95
0.016
miR-21HiCancer/Low
4.25
1.51–11.98
0.005
5.13
1.75–15.07
0.002
miR-21HiStroma/Low
1.53
0.91–2.55
0.100
1.61
0.96–2.70
0.068
ER−PR− HER2− (n = 105)
miR-21High/Low
2.62
1.24–5.55
0.010
3.29
1.47–7.37
0.003
miR-21HiCancer/Low
2.25
0.29–17.56
0.431
2.71
0.32–22.71
0.338
miR-21HiStroma/Low
2.66
1.23–5.77
0.011
3.37
1.45–7.81
0.004
Data are given as HRs and their 95% CIs for miR-21 expression and standard clinical indicators using Cox proportional hazard models. The following variables were adjusted for in multivariate analyses: age, tumor size, grade, and lymph node involvement. Risks associated with miR-21High score are reported as a whole group (miR-21High) and as subgroups (miR-21HiCancer and miR-21HiStroma) divided by tumor compartment of expression.
Tumor Compartment–Specific Expression of miR-21 Defines Risk in Distinct Molecular Subtypes
To assess the contribution of tumor compartment of miR-21 expression to the risk associated with disease outcome, the miR-21High group was subdivided into a miR-21HiCancer subgroup of 20 cases, which included only cases with tumor epithelia–specific expression, and a miR-21HiStroma group of 187 cases with predominant expression in the tumor stroma. The miR-21HiCancer subgroup had a higher risk for shorter RFS and CSS (Tables 2 and 3 and Supplemental Tables S4 and S5) than the miR-21HiStroma subgroup relative to the miR-21Low group. Because the ER/PR/HER2 status of breast cancer tumors drives their biological features and evolution,
Subtyping of breast cancer by immunohistochemistry to investigate a relationship between subtype and short and long term survival: a collaborative analysis of data for 10,159 cases from 12 studies.
we determined if ER/PR/HER2 status influenced the tumor compartment–specific effects of altered miR-21 expression. When cases were stratified by the status of individual receptors, poor clinical outcome for the miR-21HiCancer subgroup was only statistically significant in ER+ cases or in PR+ cases, and for the miR-21HiStroma subgroup, in ER− cases or PR− cases (Supplemental Tables S4 and S5). In addition, poor clinical outcome associated with the miR-21HiCancer and miR-21HiStroma subgroups was statistically significant only for HER2− cases (Figures 2 and 3 and Supplemental Tables S4 and S5). When cases were stratified by the combined status of ER/PR/HER2 as molecular surrogate of major gene expression subtypes,
poor clinical outcome associated with the miR-21HiCancer subgroup in ER+ and/or PR+HER2− cases (5-year CSS: HR, 7.05; P = 0.005). The miR-21HiStroma subgroup (Figures 2 and 3 and Tables 2 and 3) was associated with poor outcome in ER−PR−HER2− cases (5-year CSS: HR, 3.09; P = 0.017).
Figure 2Kaplan-Meier plots of disease recurrence on the basis of miR-21 expression. Kaplan-Meier survival curves for breast cancer–specific recurrence were plotted by miR-21 category for all cases or major subtypes on the basis of ER/PR/HER2 status. Curves were also plotted by tumor compartment (miR-21HiCancer, miR-21HiStroma, and miR-21Low).
Figure 3Kaplan-Meier plots for disease outcome on the basis of miR-21 expression. Kaplan-Meier survival curves for breast cancer–specific deaths were plotted by miR-21 category for all cases or major subtypes on the basis of ER/PR/HER2 status. Curves were also plotted by tumor compartment (miR-21HiCancer, miR-21HiStroma, and miR-21Low).
miRNA diagnostics is an emerging area of novel prognostic and predictive indicators for BC. Tissue slide–based assays are routine procedures in clinical laboratories to diagnose, prognosticate, and guide treatment selection. To facilitate clinical integration of ancillary miRNA detection assays, we implemented a tissue slide–based fully automated staining assay in a Clinical Laboratory Improvement Amendments–certified environment. Herein, we used this multiplex fluorescent assay to determine molecular and cellular characteristics of BC tumors by measuring the tumor compartment and levels of miR-21 expression. In this biomarker discovery study, we found that contextual expression of miR-21 provided clinically relevant information.
We determined altered expression of miR-21 at single-cell resolution and extracted this contextual information from the tumor epithelia and stroma (Figure 1). This large retrospective study in a patient cohort of 901 early-stage BC cases indicated that both expression levels and compartment-specific expression of miR-21 contained prognostic information. This information was independent of standard clinicopathological characteristics (Tables 2 and 3), including tumor grade, which is indicative of the differentiation status and proliferative rate of cancer cells. Tumor grade is an important factor in prognostic tools, such as the Nottingham Prognostic Index and Adjuvant! Online.
The prognostic value of grade is limited in ER− cases (HER2-overexpressing and TNBC subtypes) because these tumors frequently have a high grade (grades 2/3).
Our results suggest that a combination of miR-21 score (tumor stroma characteristic) and grade (tumor epithelia characteristic) may be useful for identifying tumors having more aggressive features.
By using RNA extracted from whole tissue biopsy specimens, altered expression of miR-21 has been frequently observed in BC and other cancer types.
Sempere LF, Kauppinen S: Translational Implications of MicroRNAs in Clinical Diagnostics and Therapeutics: Handbook of Cell Signaling. Edited by RA Bradshaw, EA Dennis. Oxford, Academic Press, 2009, pp 2965–2981
Implicit in these findings is the idea that miR-21–mediated regulation is confined to cancer cell–specific processes. We identified a small subgroup of cases with high miR-21 expression exclusively within the tumor epithelia (Figure 1) that were associated with poor clinical outcome (Figures 2 and 3). However, our results indicate that most BC tumors up-regulate miR-21 expression in the tumor stroma (Figure 1). Thus, miR-21–mediated regulation of biological processes in the reactive tumor stroma is likely a more frequent mechanism by which miR-21 affects BC tumor biological features. The fact that high levels of expression in the tumor stroma were associated with poor disease outcome in ER−PR−HER2−/TNBC cases (Figures 2 and 3) suggests that the intrinsic biological features of different subtypes contextualizes the effect and influence of miR-21 activity. Consequently, miR-21 expression levels, tumor compartment of miR-21 expression, and molecular characteristics (eg, ER/PR/HER2 status) appear to contribute to the biological effects of miR-21 and its value as a prognostic indicator and potential therapeutic target in BC.
The association between miR-21 expression and TNBC subtype is highly relevant. There are currently a few specific prognostic markers and no effective targeted therapies for TNBC.
Seminal studies using gene expression profiling identified an aggressive basal-like subtype, which was highly enriched for TNBC cases, on the basis of intrinsic cancer cell characteristics.
More recently, systems biology and integrative pathway analysis approaches have defined gene expression signatures that classify TNBC into four to six subgroups.
Stromal expression of mesenchymal cells and infiltrating immune cells are main attributes of some of these subgroups. It will be important to determine whether stromal expression of miR-21 correlates with one of more of these TNBC subgroups with tumorigenic stromal features or is an independent risk factor.
We previously showed that miR-21 was expressed in tumor stromal cells that also expressed smooth muscle actin and vimentin in BC tissues.
Although we did not costain with these (myo)fibroblast markers in this study, the morphological features of miR-21–expressing cells were consistent with those of tumor-associated fibroblasts.
miR-21 expression in tumor stromal fibroblast-like cells has been recently associated with poor clinical outcome in colon and pancreatic cancers.
Moreover, in vitro co-culture studies of cancer cell lines and fibroblasts (either from established cell lines or freshly derived from patients) indicate that miR-21 influences fibrogenic processes that enhance cancer cell aggressiveness and invasion.
In the colon cancer co-culture model, the reversion-inducing cysteine-rich protein with kazal motifs, a negative regulator of prometastatic matrix metalloprotease 2, was identified as a key target of miR-21.
In cardiac and/or renal mouse models of injury-induced fibrosis, key targets of miR-21 were identified as sprouty homolog 1/2 of Drosophila gene, programmed cell death 4, peroxisome proliferator–activated receptor α, and phosphatase and tensin homolog.
suggesting that there is at least some overlap of miR-21 activity in different cell types and diseases. This knowledge could be exploited to implement tumor compartment–specific anti–miR-21–based therapies in BC, especially in TNBC cases for which effective targeted therapies are still lacking.
It is an intriguing possibility that altered miR-21 expression may cause some of these morphological manifestations. We determined contextual miR-21 expression and generated a miR-21 score on the basis of tissue core stains (Figure 1). Although we discarded tissue cores that did not contain tumor lesions, it is possible that not all cores that we analyzed provided an optimal sampling of the entire tumor. Patients in this study cohort were diagnosed with BC between 1985 and 1997 and were treated in different health care centers. Treatments varied: 47.4% of patients received only local therapy, and 52.6% of patients also received adjuvant treatment (hormone therapy and/or chemotherapy) (Supplemental Table S1). Adjuvant treatments did not match current protocols, namely, use of third-generation aromatase inhibitors (eg, exemestane and anastrozole) for ER+ cases, anti–HER2-targeted therapies (eg, herceptin) for HER2-overexpressing cases, and modifications in chemotherapy combinations and modalities for specific patient subtypes and subgroups. Thus, one should be cautious when interpreting the prognostic value of altered miR-21 expression due to diverse treatment histories. An important first step to address these caveats and to pursue clinical implementation of a tissue slide–based miR-21 detection assay will be to validate these findings in whole-tissue specimens via multi-institutional clinical studies. Future studies are also needed to investigate whether miR-21 expression could serve as a predictive indicator of treatment response in clinical trials evaluating specific treatments. Such studies would reinforce the concept that miR-21 activity in the TME is biologically relevant in BC.
In conclusion, this study provides both a promising miRNA prognostic biomarker and a robust method to bring tissue slide–based miRNA detection assays closer to routine clinical practice. If these findings are supported and validated by independent clinical studies, the tumor compartment and level of miR-21 expression could become useful ancillary indicators for risk stratification in specific BC subtypes or their subgroups.
Acknowledgments
We thank Carol Valentine, Rebecca O'Meara, and Eric York (Dartmouth Pathology Translational Research Laboratory) for their technical support and assistance; Drs. Angeline Andrew, Elena Bryleva, Andrew DeCastro, Arti Gaur, and Jennifer Westerhuis for critical reading of the manuscript; and the NCI Cancer Diagnosis Program for providing TMA slides. (Because of the nature of the program, other investigators may receive and analyze slides from these same array blocks.)
Multiviewer quantification of miR-21 expression. L.F.S. and W.A.W. independently scored digital images of 399 representative cases using the same categories (1 to 4) for intensity of miR-21 stain as in Figure 1. Computer-assisted (CA) digital analysis of the same 399 images rendered scores of 1, 3, and 4 for histogram-based segmented images with <1%, >1%, and >1.5% of the combined pixels for miR-21 signal, respectively, which were located in the 3 and 4+ fold over background categories. Consensus score was the arithmetic average of ocular (L.F.S.), digital (L.F.S. or W.A.W.), and CA digital scores. Agreement between initial scores from different viewers was measured with κ coefficients. After group review and discussion, scores of one viewer (W.A.W. digital), who had applied a more stringent criterion to assign a score of miR-21High, became more similar to those of other viewers (data not shown). Ultimately, cases with a score of 1 to 2 and 3 to 4 received a miR-21 score of low and high, respectively.
Mammostrat as an immunohistochemical multigene assay for prediction of early relapse risk in the tamoxifen versus exemestane adjuvant multicenter trial pathology study.
Subtyping of breast cancer by immunohistochemistry to investigate a relationship between subtype and short and long term survival: a collaborative analysis of data for 10,159 cases from 12 studies.
Sempere LF, Kauppinen S: Translational Implications of MicroRNAs in Clinical Diagnostics and Therapeutics: Handbook of Cell Signaling. Edited by RA Bradshaw, EA Dennis. Oxford, Academic Press, 2009, pp 2965–2981
Supported by National Cancer Institute grants R03 CA141564 and R21 CA141017 (L.F.S.), National Center for Research Resources grant R21 RR024411 (W.A.W.), Van Andel Research Institute research funds (L.F.S.), a Hitchcock Foundation pilot grant (L.F.S.), and generous donations to the VAI Purple Community fund to support breast cancer research (L.F.S).
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