Background
Methods
Study objectives
Data acquisition and preprocessing
Unsupervised analysis of the tumor samples
Sample cluster enrichment and survival analysis
Analysis of differentially expressed genes and gene enrichment
Analysis of differentially methylated CpGs, correlation to expression and CpG enrichment
Results
Separation of luminal-A and luminal-B samples is not reconstructed by RNA-Seq unsupervised analysis
Unsupervised partition of luminal samples predicts survival and recurrence better than PAM50
Luminal-A samples have two distinct classes exhibiting clinical significance
Group characteristic | LumA-R1 | LumA-R2 |
P value |
---|---|---|---|
Recurrence-free survival | Increased recurrence | Reduced recurrence | 7.6e-3 |
Histological type | Ductal (p = 2.1e-05) | Lobular (p = 9.7e-12) | |
Age, years, average | 61.5 | 57.4 | 2.6e-05 |
Proliferation score | -0.4 | -0.6 | 8.9e-25 |
Tumor nuclei percent | 80 % | 73 % | 2.6e-12 |
Normal cell percent | 2.9 % | 6.1 % | 2.8e-08 |
Gene overexpression | 194 | 1068 |
Luminal-A subgroups exhibit distinct immune system expression profiles
Enrichment type | Term | Number of genes |
P value |
---|---|---|---|
Gene Ontology | Regulation of immune system process | 152 | 3.74e-50 |
Immune system process | 201 | 3.65e-47 | |
Regulation of leukocyte activation | 71 | 2.37e-28 | |
Regulation of multicellular organismal process | 183 | 2.89e-28 | |
Cell activation | 91 | 4.59e-28 | |
Regulation of response to external | 73 | 8.18e-27 | |
Regulation of biological quality | 218 | 1.82e-26 | |
Leukocyte activation | 67 | 1.95e-26 | |
Positive regulation of cell activation | 56 | 5.13e-24 | |
T cell activation | 45 | 4.93e-22 | |
Regulation of cell proliferation | 128 | 1.83e-21 | |
KEGG Pathways | Cytokine-cytokine receptor interaction | 56 | 4.76e-22 |
Hematopoietic cell lineage | 29 | 1.50e-17 | |
Cell adhesion molecules (CAMs) | 30 | 4.08e-13 | |
Primary immunodeficiency | 16 | 8.70e-13 | |
Chemokine signaling pathway | 31 | 1.14e-09 | |
Complement and coagulation cascades | 17 | 1.36e-08 | |
T cell receptor signaling pathway | 20 | 1.30e-07 | |
Allograft rejection | 11 | 6.44e-07 | |
Natural killer cell mediated cytotoxicity | 20 | 5.66e-06 | |
Pathways in cancer | 34 | 1.49e-05 | |
Wiki-Pathways | TCR signaling pathway | 10 | 1.55e-09 |
B cell receptor signaling pathway | 10 | 1.72e-06 | |
Focal adhesion | 11 | 5.88e-05 | |
Complement activation, classical pathway | 6 | 8.38e-05 | |
Chromosomal location | 11q23 | 18 | 1.84e-05 |
Xq23 | 8 | 4.99e-05 |
Analysis of DNA methylation identifies a luminal subgroup characterized by hyper-methylation and a significantly poorer outcome
Analysis of differentially methylated CpGs between the LumA-M1 and LumA-M3 subgroups and their correlation to gene expression
(1) | (2) | (3) | ||||
---|---|---|---|---|---|---|
Hyper-methylated CpGs | Negative: R < -0.2 | Positive: R > 0.2 | ||||
1000 CpGs, 483 genes | 586 CpGs, 340 genes | 212 CpGs, 125 genes | ||||
Term |
P value | Term |
P value | Term |
P value | |
Gene Ontology | Anatomical structure development | 6.1e-28 | Developmental process | 7.8e-06 | Pattern specification process | 1.1e-13 |
Developmental process | 2.0e-25 | Single organism signaling | 2.4e-05 | Regionalization | 1.1e-12 | |
Multicellular organismal process | 9.6e-24 | Signaling | 1.8e-05 | Anatomical structure development | 2.2e-11 | |
Single multicellular organism process | 1.6e-22 | Cellular developmental process | 1.4e-05 | Single organism developmental process | 1.9e-11 | |
Single organism signaling | 1.7e-21 | Single organism developmental process | 2.3e-05 | Anatomical structure morphogenesis | 1.8e-11 | |
Signaling | 1.9e-21 | Anatomical structure development | 8.0e-05 | Developmental process | 1.7e-11 | |
Cell-cell signaling | 1.7e-21 | Cell-cell signaling | 1.8e-04 | Embryonic morphogenesis | 1.1e-10 | |
Neuron differentiation | 1.2e-20 | Cell differentiation | 2.2e-04 | Cellular developmental process | 1.8e-10 | |
Single organism developmental process | 1.4e-19 | Synaptic transmission | 4.4e-04 | Organ development | 5.3e-10 | |
Regulation of transcription from RNA polymerase II promoter | 1.2e-16 | Anatomical structure morphogenesis | 6.1e-04 | Single multicellular organism process | 5.6e-10 | |
InterPro | Homeobox | 3.6e-35 | Homeobox | 1.1e-04 | Homeobox | 2.1e-31 |
Tumor suppressor genes (TSGene 2.0) | AHRR, AKR1B1, BMP2, C2orf40, CDH4, CDO1, CDX2, CNTNAP2, CSMD1, DLK1, DSC3, EBF3, EDNRB, FAT4, FOXA2, FOXC1, GALR1, GREM1, GRIN2A, ID4, IRF4, IRX1, LHX4, MAL, MIR124-2, MIR124-3, MIR125B1, MIR129-2, MIR137, MIR9-3, ONECUT1, OPCML, PAX5, PAX6, PCDH8, PHOX2A, PRKCB, PROX1, PTGDR, RASL10B, SFRP1, SFRP2, SHISA3, SLIT2, SOX7, TBX5, UNC5D, ZIC1 | 1.5e-03 | AKR1B1, ASCL1, BIN1, BMP4, CCDC67, CDK6, CDO1, EBF3, GSTP1, ID4, IRX1, L3MBTL4, LRRC4, MAP4K1, MME, NTRK3, PCDH10, PDLIM4, PROX1, PTGDR, RUNX3, SCGB3A1, SFRP1, SLC5A8, SLIT2, UBE2QL1, UNC5B, VIM, WT1 | 9.7e-02 | AMH, GATA4, HOPX, HOXB13, LHX4, LHX6, MAP4K1, ONECUT1, PAX5, RASAL1, TBX5, TP73, WT1, ZIC1 | 5.5e-02 |
(48 genes) | (29 genes) | (14 genes) |
Cox survival analysis
Survival | Recurrence | |||
---|---|---|---|---|
Variable | Hazard ratio |
P value | Hazard ratio |
P value |
LumA-R (1 vs 2) | 0.56 | 0.36991 |
0.06
|
0.00693
|
LumA-M (2, 3 vs 1) |
6.68
|
0.00484
| 3.04 | 0.07028 |
Age (<60 vs > =60 years) |
11.20
|
0.0037
| 1.03 | 0.96530 |
Pathologic stage (I, II vs. III, IV) | 2.12 | 0.25519 | 1.93 | 0.26992 |
ER status | 7.17 | 0.18095 | 0.00 | 0.99575 |
PR status | 0.47 | 0.50039 | 0.29 | 0.29092 |
Her2 status | 1.48 | 0.72659 | 0.64 | 0.68789 |