Table S1:Additional Tissue Cohorts for Validation

Table S1:Additional tissue cohorts for validation.

Samples / Sample Number / Format
FFPE Tissue Samples used for WG-DASL and qRT-PCR / IDC-DCIS Cohort (Table 1) / 17
Normal (RM-NS)a / 3
Total / 20 / RNA
FFPE Tissue Samples used for qRT-PCR / IDC-DCIS Cohort (Table 1) / 16
Normal (RM-NS) / 20
Total / 36 / RNA
FFPE Tissue Samples used for SOX10 IHCb / TMA 1: IDC NST with/without DCIS / 45
- Grade 1 / 6
- Grade 2 / 19
- Grade 3 / 20
TMA 1: DCIS / 11
TMA 2: ILC / 49
- Grade 1 / 2
- Grade 2 / 41
- Grade 3 / 6
Total / 105 / TMA
Fresh-Frozen Tissue Samples used for SFRP1 IHC
- Normal Breast only / 4
-DCIS with (2) /without (3) IDC / 5
- Invasive Carcinoma / 14
Total / 23 / Whole section
Fresh-Frozen Tissue Samples used for COL11A1 IF / Normal (RM) / 5
IDC and DCIS (+/- normal) / 5
- IDC NST / 5
- DCIS with/without IDC / 3
Total / 10 / Whole section
a Reduction Mammoplasties were carefully assessed to exclude samples showing inflammation and fibrocystic change.
bTissue Microarrays (TMAs) were constructed using a manual Tissue Arrayer (MTA-1, Beecher Instruments and Estigen Tissue Science) and 0.6 mm needles (Beecher Instruments, Cat No MP06). Scoring was performed on duplicate tissue cores and the final score was obtained from the average of the replicates or alternatively, from a single core when the replicate was not available. Any positive staining localised to the nucleus of the tumour epithelial cells of IDC and DCIS was regarded as positive expression for SOX10.

Table S2:Immunohistochemistry data for SFRP1 in normal epithelial cells, DCIS and invasive carcinomas.

Case ID / Normal TDLU / DCIS / Invasive Cancer
% / Intensity / Grade / % / Intensity / Type / Grade / % / Intensity
Q4 / 50 / 2-3+
Q19 / 20 / 2+
Q78 / 100 / 3+
Q161 / 100 / 2-3+
Q16 / 60 / 2+ / High / 30% / 1+
Q52 / High / 10% / 1+
Q106 / 100 / 2-3+ / Intermediate / 40% / 1+
Q272 / Intermediate / 20% / 1+ / IDC NST / 2 / 20 / 1+
Q25 / 30 / 2-3+ / Intermediate / 40% / 1+ / Tubular / 1 / 30% / 1+
Q27 / 100 / 3+ / IDC NST / 3 / 10% / 2+
Q46 / 100 / 3+ / IDC NST / 3 / 50% / 1+
Q48 / Metaplastic / 3 / 60% / 1 / 2+
Q83 / 60 / 2+ / IDC NST / 3 / 0
Q84 / 100 / 3+ / ILC / 2 / 40 / 1+
Q90 / 50 / 3+ / IDC NST / 3 / 20 / 1+
Q93 / 100 / 3+ / IDC NST / 2 / 40 / 1+
Q118 / 100 / 3+ / IDC NST / 3 / 10 / 1+
Q137 / IDC NST / 2 / 0
Q154 / 100 / 2-3+ / IDC NST / 2 / 30 / 1+
Q248 / Mixed Ductolobular / 2 / 20 / 1+
Q267 / Mixed Ductolobular / 3 / 50 / 2+

%, percentage of cells staining; IDC NST, invasive ductal carcinoma no special type; ILC, invasive lobular carcinoma.

Cells featuring a strikethrough represent cases for which that specific sample type was not available. Statistical analysis (performed in GraphPad Prism 5.0c), comparing median percentage expression between paired compartments and using Fisher’s Exact Test: TDLU vs. DCIS = P<0.001; TDLU vs. invasive carcinoma = P<0.001; DCIS vs. invasive carcinoma = not significant; invasive carcinomas: IDC NST vs. special types (tubular, ILC, mixed): P= 0.003.

Table S3: List of GO terms attributed to the genes differentially expressed between DCIS and IDC (n = 58 genes; this study) and between DCIS and IDC (n=546; [1]), using a significance value cut-off of P<0.05.

DCIS vs. IDC (n = 58; this study) / DCIS vs. IDC (n=546; Schuetz etal [1])
GO Term / P value / GO Term / P value
Phosphate transport / 0.0002 / Proteinaceous extracellular matrix / 2.10E-28
Tissue development / 0.0029 / Extracellular matrix / 3.77E-28
Inorganic anion transport / 0.0030 / Extracellularregion / 6.28E-25
Organ development / 0.0031 / Extracellular region part / 1.45E-22
Multicellular organismal process / 0.0037 / Extracellular matrix part / 1.61E-21
System development / 0.0037 / Extracellular matrix structural constituent / 3.38E-15
Ectoderm development / 0.0037 / Multicellular organismal process / 1.63E-11
Collagen / 0.0040 / Response to stimulus / 1.35E-10
Anion transport / 0.0040 / Immune system process / 1.35E-10
Anatomical structure development / 0.0048 / Immune response / 9.50E-10
Proteinaceous extracellular matrix / 0.0050 / Cell adhesion / 1.22E-09
Extracellular matrix / 0.0056 / Biological adhesion / 1.22E-09
Epidermis development / 0.0237 / Phosphate transport / 1.22E-09
Multicellular organismal development / 0.0267 / Structural molecule activity / 2.07E-06
Extracellular region part / 0.0482 / Response to wounding / 6.53E-06
Inorganic anion transport / 9.49E-06
Response to external stimulus / 2.05E-05
Anion transport / 2.92E-05
Multicellular organismal development / 1.46E-04
Developmental process / 0.0025
Plasma membrane / 0.003
Intrinsic to plasma membrane / 0.0038
Extracellular space / 0.0041
Response to stress / 0.0041
Integral to plasma membrane / 0.0058
Calcium ion binding / 0.0058
Plasma membrane part / 0.0205
Cellularcomponent / 0.027

Online IHC and IF Methods

IHC for SFRP1 (1:200; 1 hour incubation at room temperature; clone ab4193; Abnova, Taipei City, Taiwan) and SOX10 (1:200; 1 hour at room temperature; clone N-20; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) was performed on FF and FFPE sections, respectively. Tissue sections (4 µm) from fresh-frozen samples were cut and kept at -80C until required.Antigen retrieval using citrate buffer was only required for SOX10. The MACH 1 Universal HRP-Polymer kit (Biocare Medical, Concord, CA,USA) was used for detection;DAB chromogenic staining was prepared by mixing 30 l of DAB Chromogen in 1 ml of DAB Substrate Buffer and sections incubated for 10 min. Sections were then rinsed with deionized water and counterstained with Haematoxylin.Normal breast tissue was used as positive control for both antibodies. Positive expression for SOX10 is observed in the nuclei of the myoepithelial cells [2], whereas immunostaining for SFRP1 is found in the cytoplasm of thenormal epithelial cells [3,4]. Negative controls, where the primary antibody was omitted and only the secondary antibodies were included, were performed for each run.

Dual IF for COL11A1 (1:50, overnight incubation at 4°C; clone H-179, Santa Cruz Biotechnology Inc) and CK8/18 (1:100, 1 hour incubation; clone 5D-3, Novocastra, Leica Microsystems Pty Ltd, North Ryde, NSW, Australia) was performed on methanol fixed frozen tissue sections.Secondary antibodies (goat anti rabbit AlexaFluor488IgG (1:300 dilution) and goat anti-mouse IgG1-γ1AlexaFluor594 (1:400); Invitrogen, Mulgrave, Vic, Australia) were incubated for 30 minutes and DAPI was used for nuclear counterstaining. Slides were imaged using a confocal microscope (Carl Zeiss Pty Ltd, North Ryde, NSW, Australia) and analysed using Volocity 5.1.1 (PerkinElmer, Glen Waverley, VIC, Australia). Normal breast tissue was used as the positive control. Negative controls, where the primary antibody was omitted and only the secondary antibodies were included, were performed for each run.

Staining intensity was compared between epithelial and adjacent stromalcompartments using two methods. A pathologist (ACV) manually separated the two compartments into i) Regions of Interest (ROI) or ii) defined 50 individual points within each compartment. The software calculated the signal intensity of each ‘ROI’ or ‘point’ and a mean ROI or point calculated per compartment within each sample.Mann-Whitney U test was used for statistical analysis.

Figure S1:Dual Immunofluorescence (IF) for COL11A1 and CK8/18

Regions of Interest (ROI) separating the epithelial (A) and stromal (B) breast compartments; point by point imaging from epithelium (C) and stroma (D). Scale is 100 m.

FigureS2:ROI vs. Point by Point Analysis of COL11A1 expression in Cancer and RM samples.

Mean Signal Intensity for COL11A1 (Alexa Fluor 488) displayed as ROI (A) and Point by point analysis (C). Mean signal intensity of ROIs for CK8/18 (Alexa Fluor 594, B). Note that the ROI and Points analysis of COL11A1 replicate well ((A) and (C)). Similarly high levels were observed in the normal epithelium (NE) from both breast cancer (BC-NE) and healthy patients (RM-NE; mean signal intensity, point analysis: 74.7 and 63.9, respectively, data not shown). COL11A1 expression was higher in IDC compared to DCIS (mean signal intensity, point analysis; 73.9 vs 51.5, respectively) and expressionlevels are lower still in all types of stroma (IDC-S, DCIS-S, NB-S; meansignal intensity: 32.4,21.3 and28.5 respectively). The ROI analysis of CK8/18 is also an internal control to validate the approach, as expected, CK8/18 is exclusively detected in the epithelial samples. NB-E: Normal breast epithelium from both BC-NE and RM-NE; NB-S: Normal breast epithelium from both BC-NS and RM-NS.

Figure S3: Pairwise sample correlations of five technical replicates from WG-DASL.

Sample pairs are: 4801489029_C – 4804882009_F; 4804882017_E – 4804882008_G; 4804882011_D – 4801489028_E; 4801489029_A – 4804882018_D; 4804882009_A – 4804882008_F. Note correlations are between 0.89-0.97. Comparisons performed post normalisation.

Figure S4: Supervised K-means clustering of epithelial samples

(A) k=2 and (B) k=3 based K-means clustering analysis of the epithelial sample cohort using the 64 probes (58 genes) that were differentially expressed between DCIS and IDC (IDC grade 3, purple; IDC grade 2, pale blue; DCIS high-grade, yellow; DCIS intermediate (Int) grade, pink). Data were quantile normalised and the clustering was performed in Genespring GX 12.0 using Euclidean distance and 50 iterations. Expression level is as detailed in the colour range legend.

Figure S5:Meta-analysis of SFRP1 expression in three publically available gene expression profiling datasets (Jonsson et al.,[5]), NKI [6]) and The Cancer Genome Atlas (TGCA, level 2 gene expression data (log2 lowess normalized)). Datasets were stratified into molecular subtype [7], and an analysis of variance (ANOVA) was employed to determine any differential gene expression of SFRP1according to the breast molecular subtypes. A Tukey (multiple comparison)post hoc test was used to detect the difference between each pair of subtypesand showed that SFRP1 was significantly overexpressed in Basal breast cancer (the molecular subtype encompassing most Triple Negative cancers) compared to other molecular subtypes (Lum A, luminal A; Lum B, luminal B and HER2) in all datasets (P≤5e-05). This analysis was performed in R version 2.10.1 (

Figure S6:IF co-staining for COL11A1 in stromal fibroblasts and normal epithelium (RM).

Fibroblasts displayed positive staining with COL11A1 (arrowhead) but not with CK8/18 (A), which confirmed the mesenchymal nature of these positive cells. In contrast, normal epithelium (B) co-stained with both COL11A1 (arrow) and CK8/19. A stromal fibroblast (arrowhead) is displayed adjacent to the epithelium. DAPI counterstain in blue; COL11A1 is green (Alexa Fluor 488) and CK8/18 is red (Alexa Fluor 594). Merged image is displayed in the right panel.

References

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