Background
Breast cancer (BC) begins as premalignant lesions, progressing to the preinvasive stage of ductal carcinoma in situ (DCIS) and culminating as invasive ductal carcinoma (IDC) [
1,
2]. DCIS represents 20–25% of newly diagnosed BC and up to 40% can progress to IDC [
3]. Gene expression profiling-based studies have shown that distinct stages of progression are evolutionary products of same clonal origin and that genes conferring invasive growth are disrupted during preinvasive stages [
4‐
8]. Differences among these stages are not clear and there is no consensus as to how gene activation or inactivation alters the course of BC progression.
DCIS is a form of BC where epithelial cells restricted to the ducts exhibit an atypical phenotype [
8]. Interestingly, some DCIS lesions progress to IDCs, while others remain unchanged [
9]. Finding gene expression patterns that could predict invasive progression would allow us to personalize DCIS treatment to each patient’s real needs.
In this study, gene expression profiling was performed in non-neoplastic breast epithelium, pure DCIS, mixed lesions (DCIS-IDC) (IDC with an in-situ component) and pure IDCs, aiming to identify molecular predictors of invasive disease risk.
Materials and methods
Study population
Formalin-fixed paraffin-embedded (FFPE) breast blocks of 3 healthy women were selected as non-neoplastic breast epithelium. Specimens with pathological lesions (IDC, DCIS, DCIS-IDC) were obtained from the Department of Pathology of Barretos Cancer Hospital-Sao Paulo, Brazil. Archival FFPE blocks from 6 patients diagnosed with IDC, 6 with DCIS and 6 with IDC with in-situ (DCIS-IDC) component were selected (Table
1). Cases of IDC and DCIS-IDC were chosen considering the molecular subtype, according to St. Gallen consensus [
13]. Pathological staging was defined by current edition in 2015 of TNM classification [
10]. Histological grade was determined as Lakhani et al. [
12]. Myriad’s hereditary cancer tests were done by Myriad Genetic Laboratories, Inc. (
Salt Lake City, Utah, USA) through observations of deleterious mutations, as published by Frank et al. [
11]. Selected patients had a mean age of 55 years and were not under risk of hereditary BC, they did not present metastasis and did not receive any treatment prior surgery.
Table 1Patients characteristics
G1P1 | 50–60 | IDCa | Luminal B Her negativo | IDC | 3 | 3 | N | N | N | N | 31 | T2 | N0 | IIa | N > 50 |
G1P2 | 50–60 | IDC | TNd | IDC | 3 | 3 | N | N | N | N | 10 | T1b | N0 | I | N > 50 |
G1P3 | 60–70 | IDC | HER2 | IDC | 3 | 3 | N | N | N | N | 33 | T2 | N1 | IIb | N > 50 |
G1P4 | 60–70 | IDC | Luminal A | IDC | 2 | 2 | N | N | N | N | 20 | T1c | N0 | I | N > 50 |
G1P5 | 40–50 | IDC | Luminal B Her positivo | IDC | 1 | 1 | N | N | N | N | 40 | T2 | N0 | IIa | N < 50 |
G1P6 | 40–50 | IDC | Luminal B Her negativo | IDC | 2 | 2 | N | N | N | N | 20 | T1c | N0 | I | N < 50 |
G2P1 | 70–80 | Nb | N | DCISe | N | N | 3 | S, C, M, Co | ++++ | +++ | 105 | Tis | N0 | 0 | N > 50 |
G2P2 | 40–50 | N | N | DCIS | N | N | 3 | S, C, M | – | – | 50 | Tis | N0 | 0 | N < 50 |
G2P3 | 40–50 | N | N | DCIS | N | N | 3 | S, Co, C, M | +++ | +++ | 18 | Tis | N0 | 0 | N < 50 |
G2P4 | 50–60 | N | N | DCIS | N | N | 3 | S, Co | ++++ | ++ | 30 | Tis | N0 | 0 | N > 50 |
G2P5 | 50–60 | N | N | DCIS | N | N | 3 | C, M, Co | + | + | 60 | Tis | N0 | 0 | N > 50 |
G2P6 | 50–60 | N | N | DCIS | N | N | 3 | S, M, Co | + | – | 20 | Tis | N0 | 0 | N > 50 |
G3P1 | 50–60 | *IDCc | TN | DCIS-IDC | 3 | 3 | 3 | S, C, Co | – | – | 30 | T1c | N0 | I | N > 50 |
G3P2 | 60–70 | *IDC | Her2 | DCIS-IDC | 3 | 3 | 3 | S, A, M, Co | – | – | 100 | T2 | N1 | IIb | N > 50 |
G3P3 | 50–60 | *IDC | Luminal B Her negativo | DCIS-IDC | 3 | 3 | 3 | S, C, Co | ++++ | ++ | 65 | T1c | N0 | I | N > 50 |
G3P4 | 60–70 | *IDC | Her2 | DCIS-IDC | 2 | 2 | 3 | S, C | – | – | 36 | T1a | N1 | IIa | N > 50 |
G3P5 | 30–40 | *IDC | Luminal B Her negativo | DCIS-IDC | 2 | 2 | 2 | S, C | ++++ | + | 29 | T1a | N1 | IIa | N > 50 |
G3P6 | 50–60 | *IDC | Luminal B Her positivo | DCIS-IDC | 3 | 3 | N | S, C, Co | ++++ | + | N | T1a | N0 | I | N > 50 |
Manual microdissection of epithelial cells was performed isolating the area with, at least, 70% of tumor cells. The DCIS-IDC samples were microdissected for both tissues.
Sample naming is as follows: non-neoplastic breast epithelium - control; pure IDC - IDCpure; pure DCIS - DCISpure; IDC of DCIS-IDC group - IDCcomp and DCIS of DCIS-IDC group - DCIScomp.
RNA was isolated by RecoverAll™ Total Nucleic Acid Isolation Kit (Ambion/Life Sciences, Carlsbad, California, USA), according to manufacturer’s protocols. RNAs were quantified using NanoDrop (ThermoFisher, Waltham, Massachusetts, USA) and Qubit RNA HS Assay kit (ThermoFisher).
Gene expression analysis
Multiplex gene expression analyses were performed at the Molecular Oncology Research Center-Barretos Cancer Hospital by nCounter® PanCancer Pathways panel (NanoString Technologies™, Seattle, Washington, USA), which allows the evaluation of 770 genes (730 cancer-related human genes, being 124 driver genes and 606 genes from 13 cancer-associated canonical pathways, and 40 as internal reference loci). An average of 100 ng of RNA was used for hybridization. The system analyses for gene expression digital quantification used was the nCounter® SPRINT Profiler (NanoString Technologies™).
Data analysis
Raw counts expression was analyzed using the
nSolver™ Analysis Software (NanoString Technologies™). Two-by-two comparisons were performed and differentially expressed genes (DEGs) were selected using expression levels
p-value ≤0.01. Comparisons between the noninvasive group (control and DCIS
pure), and the invasive group (IDC
pure, DCIS
comp, and IDC
comp) were performed. A heatmap comparing the 3 tissues (control, DCIS
pure, and IDC
pure) was made in
nSolver™, and a Venn diagram was constructed to select genes of interest. Gene enrichment analyses were performed by
FunRich Functional Enrichment Analysis Tool [
14], using the Gene Ontology database. Interaction network analyses were also performed at the
FunRich using FunRich database. The UALCAN [
15] was used to evaluate gene expression in BC stages available at The Cancer Genome Atlas (TCGA) database.
Discussion
Six DEGs were found in DCISpure vs DCIScomp, being 3 of them also differentially expressed between control and DCIScomp, but not between control and DCISpure. The same 3 genes (FGF2, GAS1, and SFRP1) showed distinct gene expression profiles between noninvasive and invasive groups. Thus, suggesting their involvement in DCIS progression.
Interestingly, the in-situ stage (DCIS
pure) has more molecular differences with control than the invasive stage (IDC
pure). However, considering that IDC is the most advanced stage in progression and morphology, we expected greater molecular changes in reference to non-neoplastic tissue. Our result is probably due to early acquisition of tumor enabling features, which are later followed by minor ones [
4].
DCIS
comp and IDC
comp of patients with DCIS-IDC do not have DEGs between them and are more like IDC
pure than control. Initial gene expression changes may remain necessary in DCIS-IDC since acquisition of invasive potential has not yet been completed in all cells. Also, as suggested by Muggerud et al. [
16] and Hu et al. [
17] many processes involved in DCIS progression may be expression changes in the tumor microenvironment, and not only in tumor cells [
18].
The 3 DEGs more likely involved in DCIS progression were FGF2, GAS1, and SFPR1, all downregulated in DCIScomp. This fact suggests that progression from DCISpure to DCIScomp may use silencing mechanisms more often than activating ones.
When comparing DEGs between control and DCISpure, 31% are driver genes, whereas none of the genes that may be involved in DCIS progression or DEGs between DCISpure and IDCpure is driver genes, suggesting that major alterations occur at the beginning of carcinogenesis and not at the end.
In the analysis of invasive vs noninvasive groups,
FGF2,
GAS1, and
SFPR1 were downregulated in the invasive group. Epigenetic alterations may contribute to BC progression by transcriptionally silencing specific tumor suppressor genes [
19,
20], which could explain the loss of expression that we observed.
The expression of
FGF2 was lower in BC when compared to normal tissues [
21]. In vitro assays have demonstrated a potent inhibitory effect of
FGF2 on BC cells, possibly involving MAPK cascade and cell cycle G1/S transition [
22‐
24]. Enrichment analysis has shown statistically significant interactions between
FGF2 and MAPK pathway genes and other components of the FGF family. UALCAN analysis has shown an upregulation of
FGF2 in normal tissues, in comparison to primary BC and
FGF2 downregulation is associated with tumor progression.
According to TCGA database
GAS1 is downregulated in primary breast tumors. Hedgehog (Hh) signaling has been suggested as a critical determinant of tumor progression [
25‐
28]. A progressive increase of Hh expression and Hh pathway activation has been observed from control, DCIS, DCIS with microinvasion and to IDC [
29,
30]. GAS1 protein binds Sonic hedgehog (SHH), one of three Hh proteins, and may inhibit Hh signaling [
31,
32]. The interaction of
GAS1 with
SHH was observed but was not statistically significant.
SFRP1 gene is a negative regulator of the Wnt pathway, which is aberrantly activated in BC [
33‐
35]. Statistically significant interactions of
SFRP1 with Wnt pathway genes were seen and enrichment analysis showed a negative regulation of canonical Wnt receptor signaling pathway.
SFRP1 was downregulated in primary BC in comparison to normal tissue and in invasive lesions.
Functional analyses of FGF2, GAS1 and SFRP1 suggests a role in DCIS progression, being negative regulators of cell cycle G1/S transition, Hh signaling, and the Wnt pathway, respectively. We propose that downregulation favors DCIS progression. Unfortunately, our samples could not be divided into high and low-grade DCIS, nor could we study samples according to cancer molecular subtypes. Studying these groups separately may reveal important events in the DCIS progression.
Acknowledgements
The authors thank Lucienne B. Oliveira, Histotechnical Laboratory technique in Federal University of Espirito Santo for the aid in the preparation of some slides with mammoplasties material; Ana Paula S. Louro, pathologist in immunohistochemistry laboratory of the Death Verification Service-Vitoria-Espirito Santo, for help in the selection of elective mammoplasties to be used as control; Adriane F. Evangelista, bioinformatician of Barretos Cancer Hospital, for help in delineating the bioinformatics analysis at the project beginning; Sabina B. Aleixo, oncologist at the Evangelical Hospital of Cachoeiro de Itapemirim-Espirito Santo, for aid in the initial sampling design and Adriana C. Carloni, biologist of Barretos Cancer Hospital, for help in molecular analysis.
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