Background
Methods
Patient selection and clinical specimens
Staining procedures and laser capture microdissection
Preparation of the tissue slides
Staining
Laser capture microdissection
RNA extraction and amplification
Gene expression analysis
Statistical analysis
Differential expression analysis
In silico validation
Gene set enrichment analysis
Results
Patient demographics
Patient | Age at diagnosis (years) | ER | PR | HER2 | Tumour type | Tumour grade | Maximum tumour size (cm) | pT stage | pN stage |
---|---|---|---|---|---|---|---|---|---|
6 | 27 | Neg | Neg | Neg | Ductal | 3 | 2.3 | 2 | 0 |
5 | 30 | Neg | Neg | Neg | Ductal | 3 | 2.5 | 2 | 0 |
7 | 32 | Neg | Neg | Neg | Ductal | 3 | 2.2 | 2 | 0 |
1 | 33 | Neg | Neg | Neg | Ductal | 3 | 2.8 | 2 | 0 |
3 | 39 | Neg | Neg | Neg | Ductal | 3 | 3.0 | 2 | 0 |
2 | 43 | Neg | Neg | Neg | Ductal | 3 | 3.0 | 2 | 2a |
4 | 44 | Neg | Neg | Neg | Ductal | 3 | 2.8 | 2 | 0 |
8 | 44 | Neg | Neg | Neg | Ductal | 3 | 3.5 | 2 | 0 |
9 | 44 | Neg | Neg | Neg | Ductal | 3 | 3.0 | 2 | 0 |
12 | 80 | Neg | Neg | Neg | Ductal | 3 | 4.0 | 2 | 0 |
16 | 82 | Neg | Neg | Neg | Ductal | 3 | 3.5 | 2 | 0 |
17 | 82 | Neg | Neg | Neg | Ductal | 3 | 1.5 | 1c | 0 |
13 | 82 | Neg | Neg | Neg | Ductal | 2 | 3.0 | 2 | 3a |
15 | 83 | Neg | Neg | Neg | Ductal | 3 | 3.8 | 2 | 1a |
10 | 83 | Neg | Neg | Neg | Ductal | 3 | 3.2 | 2 | 0 |
11 | 86 | Neg | Neg | Neg | Ductal | 3 | 3.0 | 2 | 0 |
14 | 87 | Neg | Neg | Neg | Ductal | 3 | 2.0 | 1c | 0 |
Differential gene expression analysis
Gene | Full name | Fold change |
---|---|---|
SPP1
| Secreted phosphoprotein 1 | −4.79 |
EPCAM
| Epithelial cell adhesion molecule | −4.02 |
IL8
| Interleukin 8 | −2.74 |
NR4A2
| Nuclear receptor subfamily 4, group A, member 2 | −2.45 |
RGS2
| Regulator of G-protein signaling 2, 24 kDa | −2.41 |
TREM1
| Triggering receptor expressed on myeloid cells 1 | −2.36 |
PROM1
| Prominin 1 | −2.27 |
SCG2
| Secretogranin II | −2.22 |
LPL
| Lipoprotein lipase | −2.20 |
SDC4
| Syndecan 4 | −2.19 |
SLC2A3
| Solute carrier family 2 (facilitated glucose transporter), member 3 | −2.13 |
PFKFB3
| 6-Phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 | −2.11 |
TNFRSF11B
| Tumour necrosis factor receptor superfamily, member 11b | −2.11 |
WIF1
| WNT inhibitory factor 1 | −2.10 |
NAMPT
| Nicotinamide phosphoribosyltransferase | −2.08 |
ENPEP
| Glutamyl aminopeptidase (aminopeptidase A) | −2.07 |
ZNF331
| Zinc finger protein 331 | −2.07 |
ANXA3
| Annexin A3 | −2.06 |
HAPLN1
| Hyaluronan and proteoglycan link protein 1 | −2.05 |
CSN3
| Casein kappa | −2.05 |
KRT23
| Keratin 23 (histone deacetylase inducible) | −2.05 |
VEGFA
| Vascular endothelial growth factor A | −2.03 |
STC1
| Stanniocalcin 1 | −2.01 |
EGLN3
| Egl nine homolog 3 (C. elegans) | −1.97 |
ADM
| Adrenomedullin | −1.96 |
G0S2
| G0/G1 switch 2 | −1.95 |
BAMBI
| BMP and activin membrane-bound inhibitor homolog (Xenopus laevis) | −1.93 |
TDO2
| Tryptophan 2,3-dioxygenase | −1.93 |
CD24
| CD24 molecule | −1.92 |
DNER
| Delta/Notch-like epidermal growth factor-related receptor | −1.92 |
IBSP
| Integrin-binding sialoprotein | −1.91 |
HSPA2
| Heat shock 70 kDa protein 2 | −1.90 |
ERRFI1
| ERBB receptor feedback inhibitor 1 | −1.89 |
MUCL1
| Mucin-like 1 | −1.89 |
APOLD1
| Apolipoprotein L domain containing 1 | −1.89 |
SHISA2
| Shisa homolog 2 (Xenopus laevis) | −1.88 |
GPX3
| Glutathione peroxidase 3 (plasma) | −1.87 |
SERPINE1
| Serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 1 | −1.87 |
COL2A1
| Collagen, type II, α 1 | −1.86 |
CP
| Ceruloplasmin (ferroxidase) | −1.85 |
COL9A3
| Collagen, type IX, α 3 | −1.85 |
ENO2
| Enolase 2 (gamma, neuronal) | −1.84 |
FOSB
| FBJ murine osteosarcoma viral oncogene homolog B | −1.84 |
TSPAN13
| Tetraspanin 13 | −1.82 |
CYP4X1
| Cytochrome P450, family 4, subfamily X, polypeptide 1 | −1.82 |
TFAP2C
| Transcription factor AP-2γ (activating enhancer binding protein 2γ) | −1.81 |
EGR3
| Early growth response 3 | −1.81 |
SOX11
| SRY (sex-determining region Y), box 11 | −1.79 |
CLEC5A
| C-type lectin domain family 5, member A | −1.78 |
CYP26B1
| Cytochrome P450, family 26, subfamily B, polypeptide 1 | −1.78 |
SLPI
| Secretory leukocyte peptidase inhibitor | −1.78 |
PI15
| Peptidase inhibitor 15 | −1.78 |
RBP7
| Retinol binding protein 7, cellular | −1.77 |
SERPINA3
| Serpin peptidase inhibitor, clade A (α-1 antiproteinase, antitrypsin), member 3 | −1.77 |
CCDC102B
| Coiled-coil domain containing 102B | −1.75 |
MTHFD2
| Methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 2, methenyltetrahydrofolate cyclohydrolase | −1.74 |
CFI
| Complement factor I | −1.74 |
FCGBP
| Fc fragment of IgG binding protein | −1.73 |
GPNMB
| Glycoprotein (transmembrane) NMB | −1.73 |
FCGR2A
| Fc fragment of IgG, low affinity IIa, receptor (CD32) | −1.72 |
MAL2
| Mal, T-cell differentiation protein 2 | −1.72 |
UAP1
| UDP-N-acteylglucosamine pyrophosphorylase 1 | −1.71 |
IER3
| Immediate early response 3 | −1.70 |
COL4A1
| Collagen, type IV, α 1 | −1.69 |
EFNB2
| Ephrin-B2 | −1.69 |
FCGR2B
| Fc fragment of IgG, low affinity IIb, receptor (CD32) | −1.69 |
BTBD3
| BTB (POZ) domain containing 3 | −1.68 |
FGF13
| Fibroblast growth factor 13 | −1.68 |
GALNT3
| UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyltransferase 3 (GalNAc-T3) | −1.67 |
INHBB
| inhibin, β B | −1.66 |
MANSC1
| MANSC domain containing 1 | −1.65 |
DSP
| Desmoplakin | −1.64 |
CLDN8
| Claudin 8 | −1.64 |
TUBB2B
| Tubulin, β 2B | −1.64 |
PODXL
| Podocalyxin-like | −1.63 |
EHF
| ETS homologous factor | −1.63 |
TIPARP
| TCDD-inducible poly(ADP-ribose) polymerase | −1.63 |
ANGPT2
| Angiopoietin 2 | −1.62 |
ADAMTS1
| ADAM metallopeptidase with thrombospondin type 1 motif, 1 | −1.62 |
GPR4
| G protein-coupled receptor 4 | −1.61 |
DBH
| Dopamine β-hydroxylase (dopamine β-monooxygenase) | −1.61 |
GPR183
| G protein-coupled receptor 183 | −1.61 |
TFAP2A
| Transcription factor AP-2 α (activating enhancer binding protein 2 α) | −1.60 |
SNORD89
| Small nucleolar RNA, C/D box 89 | −1.60 |
CXCL2
| Chemokine (C-X-C motif) ligand 2 | −1.60 |
CXADR
| Coxsackie virus and adenovirus receptor | −1.60 |
TPRKB
| TP53RK binding protein | −1.60 |
ETS2
| v-ets erythroblastosis virus E26 oncogene homolog 2 (avian) | −1.60 |
RAPH1
| Ras association (RalGDS/AF-6) and pleckstrin homology domains 1 | −1.60 |
ADGRF5
| Adhesion G protein-coupled receptor F | −1.60 |
CA2
| Carbonic anhydrase II | −1.59 |
LIPA
| Lipase A, lysosomal acid, cholesterol esterase | −1.59 |
PGM2
| Phosphoglucomutase 2 | −1.59 |
KRT19
| Keratin 19 | −1.58 |
MGAT5
| Mannosyl (α-1,6-)-glycoprotein β-1,6-N-acetyl-glucosaminyltransferase | −1.58 |
NCF2
| Neutrophil cytosolic factor 2 | −1.57 |
RHOU
| Ras homolog gene family, member U | −1.57 |
ALCAM
| Activated leukocyte cell adhesion molecule | −1.57 |
LRRN1
| Leucine-rich repeat neuronal 1 | −1.57 |
OLR1
| Oxidized low-density lipoprotein (lectin-like) receptor 1 | −1.55 |
SLC19A2
| Solute carrier family 19 (thiamine transporter), member 2 | −1.55 |
PRPS2
| Phosphoribosyl pyrophosphate synthetase 2 | −1.55 |
MEGF10
| Multiple EGF-like domains 10 | −1.55 |
CYYR1
| Cysteine/tyrosine-rich 1 | −1.54 |
PLVAP
| Plasmalemma vesicle-associated protein | −1.54 |
TM4SF1
| Transmembrane 4 L6 family member 1 | −1.54 |
PDGFA
| Platelet-derived growth factor α polypeptide | −1.54 |
YBX2
| Y box binding protein 2 | −1.54 |
ATP2B1
| ATPase, Ca2+-transporting, plasma membrane 1 | −1.54 |
PCDHB2
| Protocadherin β 2 | −1.54 |
DNMT1
| DNA (cytosine-5-)-methyltransferase 1 | −1.54 |
S100A8
| S100 calcium binding protein A8 | −1.53 |
MAP2
| Microtubule-associated protein 2 | −1.53 |
ARRDC4
| Arrestin domain containing 4 | −1.52 |
FAM83D
| Family with sequence similarity 83, member D | −1.52 |
LSR
| Lipolysis stimulated lipoprotein receptor | −1.52 |
STK26
| Serine/threonine protein kinase 26 | −1.51 |
MIR181A2HG
| MIR181A2 host gene (non-protein coding) | −1.51 |
VWA8
| von Willebrand factor A domain containing 8 | −1.51 |
MEST
| Mesoderm-specific transcript homolog (mouse) | −1.51 |
ZNF835
| Zinc finger protein 835 | 1.51 |
NAT1
|
N-acetyltransferase 1 (arylamine N-acetyltransferase) | 1.51 |
EPSTI1
| Epithelial stromal interaction 1 (breast) | 1.51 |
LOC221946
| Hypothetical LOC221946 | 1.51 |
OAS1
| 2′,5′-oligoadenylate synthetase 1, 40/46 kDa | 1.52 |
SELL
| Selectin L | 1.52 |
COX6C
| Cytochrome c oxidase subunit VIc | 1.52 |
TRIM41
| Tripartite motif-containing 41 | 1.52 |
IFI27
| Interferon-α-inducible protein 27 | 1.52 |
IGF1
| Insulin-like growth factor 1 (somatomedin C) | 1.52 |
SCAMP1-AS1
| SCAMP1 antisense RNA 1 | 1.52 |
CD207
| CD207 molecule, langerin | 1.52 |
IFI35
| Interferon-induced protein 35 | 1.52 |
GGH
| γ-Glutamyl hydrolase (conjugase, folylpolygammaglutamyl hydrolase) | 1.52 |
NOX4
| NADPH oxidase 4 | 1.53 |
CNTN3
| Contactin 3 (plasmacytoma associated) | 1.53 |
CCL5
| Chemokine (C-C motif) ligand 5 | 1.54 |
GALNT1
| UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyltransferase 1 (GalNAc-T1) | 1.54 |
SPON1
| Spondin 1, extracellular matrix protein | 1.54 |
SEMA3C
| Sema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3C | 1.54 |
DDX60L
| DEAD (Asp-Glu-Ala-Asp) box polypeptide 60-like | 1.55 |
TNFSF10
| Tumor necrosis factor (ligand) superfamily, member 10 | 1.55 |
CXCL14
| Chemokine (C-X-C motif) ligand 14 | 1.55 |
WISP2
| WNT1 inducible signaling pathway protein 2 | 1.55 |
STAT1
| Signal transducer and activator of transcription 1, 91 kDa | 1.55 |
COMP
| Cartilage oligomeric matrix protein | 1.56 |
IGLJ3
| Immunoglobulin lambda joining 3 | 1.56 |
LRRC17
| Leucine-rich repeat containing 17 | 1.56 |
IFI44
| Interferon-induced protein 44 | 1.56 |
ISG15
| ISG15 ubiquitin-like modifier | 1.56 |
FBLN2
| Fibulin 2 | 1.57 |
SLC6A6
| Solute carrier family 6 (neurotransmitter transporter, taurine), member 6 | 1.57 |
MX2
| Myxovirus (influenza virus) resistance 2 (mouse) | 1.57 |
SH3D19
| SH3 domain containing 19 | 1.57 |
TRBC1
| T-cell receptor β constant 1 | 1.58 |
SGCE
| Sarcoglycan, epsilon | 1.58 |
IGHM
| Immunoglobulin heavy constant mu | 1.58 |
DCBLD1
| Discoidin, CUB and LCCL domain containing 1 | 1.59 |
PPAPDC1A
| Phosphatidic acid phosphatase type 2 domain containing 1A | 1.59 |
BST2
| Bone marrow stromal cell antigen 2 | 1.59 |
MFAP2
| Microfibrillar-associated protein 2 | 1.60 |
PDGFD
| Platelet-derived growth factor D | 1.60 |
IGKC
| Immunoglobulin kappa constant | 1.60 |
CST1
| Cystatin SN | 1.61 |
CCL8
| Chemokine (C-C motif) ligand 8 | 1.61 |
RASGRF2
| Ras protein-specific guanine nucleotide-releasing factor 2 | 1.61 |
MX1
| Myxovirus (influenza virus) resistance 1, interferon-inducible protein p78 (mouse) | 1.63 |
PDGFRL
| Platelet-derived growth factor receptor-like | 1.63 |
ALDH1L2
| Aldehyde dehydrogenase 1 family, member L2 | 1.63 |
FAM198B
| Family with sequence similarity 198, member B | 1.63 |
MIR100HG
| Mir-100-let-7a-2 cluster host gene | 1.64 |
GAPT
| GRB2-binding adaptor protein, transmembrane | 1.65 |
SELM
| Selenoprotein M | 1.65 |
DSCAM-AS1
| DSCAM antisense RNA 1 | 1.66 |
STMN2
| Stathmin-like 2 | 1.69 |
FBLN5
| Fibulin 5 | 1.70 |
IFIT3
| Interferon-induced protein with tetratricopeptide repeats 3 | 1.70 |
SFRP4
| Secreted frizzled-related protein 4 | 1.71 |
ACKR4
| Atypical chemokine receptor 4 | 1.71 |
CPNE2
| Copine II | 1.71 |
PSMB9
| Proteasome (prosome, macropain) subunit, β type, 9 (large multifunctional peptidase 2) | 1.72 |
ST6GAL2
| ST6 β-galactosamide α-2,6-sialyltranferase 2 | 1.72 |
NEXN
| Nexilin (F actin binding protein) | 1.72 |
CD52
| CD52 molecule | 1.72 |
MFAP5
| Microfibrillar associated protein 5 | 1.73 |
RARRES3
| Retinoic acid receptor responder (tazarotene induced) 3 | 1.75 |
GXYLT2
| Glucoside xylosyltransferase 2 | 1.75 |
HMCN1
| Hemicentin 1 | 1.76 |
EFEMP1
| EGF-containing fibulin-like extracellular matrix protein 1 | 1.78 |
IL21R
| Interleukin 21 receptor | 1.78 |
C8orf4
| Chromosome 8 open reading frame 4 | 1.78 |
LINC01503
| Long intergenic non-protein coding RNA 1503 | 1.78 |
OLFML3
| Olfactomedin-like 3 | 1.79 |
CILP
| Cartilage intermediate layer protein, nucleotide pyrophosphohydrolase | 1.81 |
MVB12A
| Multivesicular body subunit 12A | 1.82 |
SCUBE2
| Signal peptide, CUB domain, EGF-like 2 | 1.83 |
WNT2
| Wingless-type MMTV integration site family member 2 | 1.85 |
APOL3
| Apolipoprotein L3 | 1.87 |
ADRA2A
| Alpha-2A adrenergic receptor | 1.89 |
HIST1H3I
| Histone cluster 1, H3i | 1.92 |
SLC46A3
| Solute carrier family 46, member 3 | 1.92 |
ARHGAP28
| Rho GTPase activating protein 28 | 1.93 |
KANK4
| KN motif and ankyrin repeat domains 4 | 1.93 |
SDC1
| Syndecan 1 | 1.95 |
CMPK2
| Cytidine monophosphate (UMP-CMP) kinase 2, mitochondrial | 1.96 |
IFI44L
| Interferon-induced protein 44-like | 1.97 |
FMO1
| Flavin containing monooxygenase 1 | 1.98 |
TMEM119
| Transmembrane protein 119 | 1.99 |
FNDC1
| Fibronectin type III domain containing 1 | 2.00 |
ADAMDEC1
| ADAM-like, decysin 1 | 2.00 |
TPSAB1
| Tryptase α/β1 | 2.02 |
CPA3
| Carboxypeptidase A3 (mast cell) | 2.02 |
MMP3
| Matrix metallopeptidase 3 (stromelysin 1, progelatinase) | 2.05 |
IFI6
| Interferon, α-inducible protein 6 | 2.06 |
IFIT1
| Interferon-induced protein with tetratricopeptide repeats 1 | 2.06 |
SFRP2
| Secreted frizzled-related protein 2 | 2.09 |
TRIM6
| Tripartite motif-containing 6 | 2.10 |
TPSB2
| Tryptase β2 (gene/pseudogene) | 2.19 |
RSAD2
| Radical S-adenosyl methionine domain containing 2 | 2.28 |
LOXL1
| Lysyl oxidase-like 1 | 2.30 |
OMD
| Osteomodulin | 2.35 |
IGJ
| Immunoglobulin J polypeptide, linker protein for immunoglobulin α and mu polypeptides | 2.44 |
FCGR1A
| Fc fragment of IgG, high affinity Ia, receptor (CD64) | 2.47 |
MATN3
| Matrilin 3 | 2.55 |
IGLV@
| Immunoglobulin lambda variable cluster | 2.65 |
OGN
| Osteoglycin | 2.99 |
EPYC
| Epiphycan | 3.04 |
Data validation
Gene | Full name | Fold change |
---|---|---|
RARRES3
| Retinoic acid receptor responder (tazarotene induced) 3 | 1.75 |
SFRP4
| Secreted frizzled-related protein 4 | 1.71 |
SCUBE2
| Signal peptide, CUB domain, EGF-like 2 | 1.83 |
NAT1
|
N-acetyltransferase 1 (arylamine N-acetyltransferase) | 1.51 |
COMP
| Cartilage oligomeric matrix protein | 1.56 |
ANXA3
| Annexin A3 | −2.06 |
PROM1
| Prominin 1 | −2.27 |
FGF13
| Fibroblast growth factor 13 | −1.68 |
TUBB2B
| Tubulin, beta 2B | −1.64 |
WIF1
| WNT inhibitory factor 1 | −2.10 |
Gene set enrichment analysis
Gene group | Involved genes | References |
---|---|---|
Cellular senescence |
CDKN1A, CDKN2A, TP53, RB1, GLB1
| |
DNA damage response |
ATM, NBN, CHEK2
| |
Senescence-associated secretory profile |
IL1A, IL6, IL6R, IL6ST, IL8, CXCL1, CXCL2, CXCL3, CSF2, IL7, ICAM1, TNFRSF11B, HGF, IGFBP4, CCL8, PLAUR, IGFBP2, CCL26, IL13, CCL20, ICAM3, PGF, TNFRSF1A, TNFRSF1B, CCL13, CCL16, TNFRSF10C, CCL2, FAS, ANG, IGFBP6, IL1B, (CCL3), TIMP2, IL11, OSM, LEP, AXL, KITLG, FGF7, IL15, FGF2, IGFBP1, MIF
| |
Autophagy-to-senescence transition |
CAV1, CTSB, BNIP3, PRKAA1, PRKAA2, LAMP2, MAP1LC3B, ATG16L1, HIF1A, NFKB1, DRAM1, TP73, MAPK8, E2F1, STK11
|