event free survival event free survival

(1)

Supplementary Table S1: Methods applied to determine the biochemical status of ER, PgR, and HER2 in the different datasets.

Biochemical Estrogen receptor status Biochem. Progesterone receptor status HER2 status

Dataset n= Method cutoff n= Method Cutoff n= Method Cutoff Reference

Rotterdam 344 LBA, EIA, (IHC n=9) 10 fmol/mg, (IHC:

10% pos. cells)

256 LBA, EIA 4 fmol/mg, n.a. n.a. n.a. 14, 15, 16

TransBIG 198 IHC not given n.a. n.a. n.a. n.a. n.a. n.a. 18

Oxford‐

Untreated

63 not given not given n.a. n.a. n.a. n.a. n.a. n.a. 19

London 87 not given not given 85 not given not given n.a. n.a. n.a. 20

London‐2 77 not given not given 77 not given not given n.a. n.a. n.a. 21

Oxford‐

Tamoxifen

109 not given not given n.a. n.a. n.a. n.a. n.a. n.a. 20

Veridex‐Tam 136 IHC, LBA 10% pos. cells, 10 fmol/mg,

n.a. n.a. n.a. n.a. n.a. n.a. 22

Frankfurt‐3 50 IHC 10% pos. tumor cells

46 IHC 10% pos. tumor

cells

19 IHC / FISH IHC3+ OR FISH >2.0 12

Uppsala 249 EIA >0.05 fmol/µg DNA 251 EIA >0.05 fmol/µg DNA n.a. n.a. n.a. 25

San Francisco 118 not given not given 117 not given not given 79 not given not given 26

New York 99 not given not given 98 not given not given 88 n.a. n.a. 27

Frankfurt 114 IHC 10% pos. tumor

cells

65 IHC / FISH IHC3+ OR FISH >2.0 10 Frankfurt‐2 65 IHC 10% pos. tumor

cells

MDA133 133 IHC 10% pos. tumor

cells

EORTC 48 IHC not given 47 IHC not given n.a. n.a. n.a. 29

Edinburgh 116 IHC not given n.a. n.a. n.a. n.a. n.a. n.a. 30

expO 153 not given not given 151 not given not given 141 not given not given 31

Boston 39 not given not given 39 not given not given 37 not given not given 34

TOTAL 2198 1474 618

(2)

Supplementary Table S2: Univariate and multivariate Cox regression analysis of disease free survival according to microarray based ER, PgR, and HER2 status among 2058 breast cancer patients.

Univariate Multivariate

Parameter n= P Value* Hazard Ratio 95 % CI P Value* Hazard Ratio 95 % CI

ER status negative vs positive 510 vs. 1548 0.002 1.34 (1.12‐1.60) 0.78 1.03 (0.84‐1.27)

PgR status negative vs positive 854 vs. 1204 <0.001 1.55 (1.32‐1.82) <0.001 1.48 (1.23‐1.78)

HER2 status positive vs negative 238 vs. 1820 0.002 1.43 (1.13‐1.80) 0.083 1.24 (0.97‐1.58)

*significant P values are given in bold

(3)

Supplementary Figure S1: Event free survival of patients according to the available endpoint.

A) Event free survival of ER positive breast cancers (n=1549) with either RFS (n=919) or DMFS (n=630) event status available.

B) Event free survival ER negative breast cancers (n=510) with either RFS (n=261) or DMFS (n=249) event status available.

months

120 100

80 60

40 20

0

event fr ee sur v ival

1.0

0.8

0.6

0.4

0.2

0.0

B

ER negative tumors (n=510)

Patients with only DMFS available (n=261) Patients with RFS available (n=249)

(P=0.18)

months

120 100

80 40 60

20 0

event fr ee sur v ival

1.0

0.8

0.6

0.4

0.2

0.0

A

Patients with only DMFS available (n=919) Patients with RFS available (n=630)

ER positive tumors (n=1549)

(P=0.25)

(4)

Supplementary Figure S2: ROC curves demonstrating the relationship of sensitivity and specifity of different Affymetrix ProbeSets for the estrogen receptor with biochemical data of the estrogen receptor status.

1 - specificity

sensitivity

Probe Set AUC 95% CI

205225_at 0.949 0.938 0.960 215552_s_at 0.820 0.802 0.839 211235_s_at 0.783 0.763 0.802 211233_x_at 0.779 0.759 0.799 217190_x_at 0.759 0.738 0.781 211234_x_at 0.752 0.731 0.772 211627_x_at 0.580 0.554 0.606 215551_at 0.533 0.508 0.558 217163_at 0.513 0.486 0.539

(5)

Supplementary Figure S3: ROC curves demonstrating the relationship between sensitivity and specificity of Affymetrix ProbeSets for the PgR (A) and HER2 genes (B) with the results from immunohistochemistry of the progesterone receptor and HER2 (3+ IHC or FISH positive).

1.0

0.8

0.6

0.4

0.2

0.0

1.0 0.8

0.6 0.2 0.4

0.0

1 - specificity

sensitivity

216836_s_at 210930_s_at

1 - specificity

sensitivity

1.0

0.8

0.6

0.4

0.2

0.0

1.0 0.6 0.8

0.4 0.0 0.2

A B

(6)

Supplementary Figure S4: Distribution of ER expression values in the individual datasets.

dataset Rotterdam Mainz TransBIG Oxford‐Untreated London

cutoff 0.0072 0.0091 0.0051 0.0034 0.0082

distribution

-0.010 -0.005 0.000 0.005 0.010 0.015 0.020

020406080100

an

-0.010 -0.005 0.000 0.005 0.010 0.015 0.020

050100150

an

-0.010 -0.005 0.000 0.005 0.010 0.015 0.020

050100150

an

-0.010 -0.005 0.000 0.005 0.010 0.015 0.020

050100150200250

an

-0.010 -0.005 0.000 0.005 0.010 0.015 0.020

0100200300

an

dataset London‐2 Oxford‐Tamoxifen Veridex‐Tam Frankfurt‐3 Stockholm

cutoff 0.0101 0.0018 0.0031 0.0040 0.0053

distribution

-0.010 -0.005 0.000 0.005 0.010 0.015 0.020

050100150200250

an

-0.010 -0.005 0.000 0.005 0.010 0.015 0.020

050100150200250300

an

-0.010 -0.005 0.000 0.005 0.010 0.015 0.020

050100150200

an

-0.010 -0.005 0.000 0.005 0.010 0.015 0.020

050100150200

an

-0.010 -0.005 0.000 0.005 0.010 0.015 0.020

020406080100120140

an

dataset Uppsala San Francisco New York Frankfurt Frankfurt‐2

cutoff 0.0092 0.0064 0.0066 0.0056 0.0064

distribution

-0.010 -0.005 0.000 0.005 0.010 0.015 0.020

050100150

an

-0.010 -0.005 0.000 0.005 0.010 0.015 0.020

050100150

an

-0.010 -0.005 0.000 0.005 0.010 0.015 0.020

020406080100120

an

-0.010 -0.005 0.000 0.005 0.010 0.015 0.020

050100150

an

-0.010 -0.005 0.000 0.005 0.010 0.015 0.020

050100150

an

(7)

dataset MDA133 EORTC Edinburgh expO Signapore

cutoff 0.0072 0.0067 0.0061 0.0080 0.0078

distribution

-0.010 -0.005 0.000 0.005 0.010 0.015 0.020

020406080100

an

-0.010 -0.005 0.000 0.005 0.010 0.015 0.020

050100150

an

-0.010 -0.005 0.000 0.005 0.010 0.015 0.020

050100150200

an

-0.010 -0.005 0.000 0.005 0.010 0.015 0.020

020406080100

an

-0.010 -0.005 0.000 0.005 0.010 0.015 0.020

020406080

an

dataset Genentech Boston combined

cutoff 0.0054 0.0093 0.0075

distribution

-0.010 -0.005 0.000 0.005 0.010 0.015 0.020

050100150200

an

-0.010 -0.005 0.000 0.005 0.010 0.015 0.020

050100150200250300

an

-0.010 -0.005 0.000 0.005 0.010 0.015 0.020

020406080100

an

(8)

Supplementary Figure S5: Distribution of PgR expression values in the individual datasets.

cutoff ‐0.0075 ‐0.0052 ‐0.0084 ‐0.0068 ‐0.0088

distribution

-0.02 -0.01 0.00 0.01

020406080100

an

-0.02 -0.01 0.00 0.01

020406080100

an

-0.02 -0.01 0.00 0.01

020406080

an

-0.02 -0.01 0.00 0.01

020406080100120

an

-0.02 -0.01 0.00 0.01

020406080100

an

cutoff ‐0.0064 ‐0.0081 ‐0.0070 ‐0.0095 ‐0.0075

distribution

-0.02 -0.01 0.00 0.01

050100150

an

-0.02 -0.01 0.00 0.01

020406080100

an

-0.02 -0.01 0.00 0.01

020406080100

an

-0.02 -0.01 0.00 0.01

020406080100120

an

-0.02 -0.01 0.00 0.01

050100150

an

cutoff ‐0.0084 ‐0.0099 ‐0.0069 ‐0.0064 ‐0.0081

distribution

-0.02 -0.01 0.00 0.01

020406080100

an

-0.02 -0.01 0.00 0.01

020406080100120140

an

-0.02 -0.01 0.00 0.01

050100150

an

-0.02 -0.01 0.00 0.01

020406080100

an

-0.02 -0.01 0.00 0.01

050100150

an

(9)

cutoff ‐0.0079 ‐0.0054 ‐0.0082 ‐0.0076 ‐0.0071

distribution

-0.02 -0.01 0.00 0.01

020406080100

an

-0.02 -0.01 0.00 0.01

050100150200

an

-0.02 -0.01 0.00 0.01

050100150

an

-0.02 -0.01 0.00 0.01

020406080100120

an

-0.02 -0.01 0.00 0.01

020406080100120

an

cutoff ‐0.0123 ‐0.0047 ‐0.0078

distribution

-0.02 -0.01 0.00 0.01

050100150200

an

-0.02 -0.01 0.00 0.01

050100150200250

an

-0.02 -0.01 0.00 0.01

020406080

an

(10)

Supplementary Figure S6: Distribution of HER2 expression values in the individual datasets.

cutoff 0.0137 0.0122 0.0124 0.0133 0.0120

distribution

-0.005 0.000 0.005 0.010 0.015 0.020 0.025

050100150

an

-0.005 0.000 0.005 0.010 0.015 0.020 0.025

050100150200250

an

-0.005 0.000 0.005 0.010 0.015 0.020 0.025

050100150200

an

-0.005 0.000 0.005 0.010 0.015 0.020 0.025

050100150200250

an

-0.005 0.000 0.005 0.010 0.015 0.020 0.025

050100150200250300

an

cutoff 0.0140 0.0120 0.0139 0.0163 0.0136

distribution

-0.005 0.000 0.005 0.010 0.015 0.020 0.025

050100150200250300

an

-0.005 0.000 0.005 0.010 0.015 0.020 0.025

050100150200250300350

an

-0.005 0.000 0.005 0.010 0.015 0.020 0.025

050100150

an

-0.005 0.000 0.005 0.010 0.015 0.020 0.025

050100150200250300

an

-0.005 0.000 0.005 0.010 0.015 0.020 0.025

050100150200250

an

cutoff 0.0135 0.0119 0.0151 0.0146 0.0145

distribution

-0.005 0.000 0.005 0.010 0.015 0.020 0.025

050100150200

an

-0.005 0.000 0.005 0.010 0.015 0.020 0.025

050100150200250

an

-0.005 0.000 0.005 0.010 0.015 0.020 0.025

050100150

an

-0.005 0.000 0.005 0.010 0.015 0.020 0.025

050100150

an

-0.005 0.000 0.005 0.010 0.015 0.020 0.025

050100150200250

an

(11)

cutoff 0.0144 0.0092 0.0121 0.0141 0.0147

distribution

-0.005 0.000 0.005 0.010 0.015 0.020 0.025

050100150

an

-0.005 0.000 0.005 0.010 0.015 0.020 0.025

050100150200

an

-0.005 0.000 0.005 0.010 0.015 0.020 0.025

050100150200250

an

-0.005 0.000 0.005 0.010 0.015 0.020 0.025

050100150

an

-0.005 0.000 0.005 0.010 0.015 0.020 0.025

020406080100120140

an

cutoff 0.0145 0.0155 0.0135

distribution

-0.005 0.000 0.005 0.010 0.015 0.020 0.025

050100150200250300

an

-0.005 0.000 0.005 0.010 0.015 0.020 0.025

050100150

an

-0.005 0.000 0.005 0.010 0.015 0.020 0.025

050100150

an

(12)

Suppl. Figure S7: Influence of gene normalization on the distribution of ER expression values.

A) Distribution of gene normalized ER expression values (ProbeSet 205225_at) in the Frankfurt dataset (n=120 samples). Two normal distributions were fitted to the data allowing separation of ER positive and ER negative samples.

B) Distribution of ER expression values after gene normalization of only ER positive samples (n=79) from the Frankfurt dataset. The same scale as in (A) has been used for the X axis.

C) Distribution of ER expression values after gene normalization of only ER negative samples (n=41) from the Frankfurt dataset. The same scale as in (A) has been used for the X axis.

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

-0.4 -0.2 0.0 0.2 0.4

Density

gene normalized ER expression ER negative samples only

0 1 2 3 4 5

-0.4 -0.2 0.0 0.2 0.4

Density

gene normalized ER expression ER positive samples only

-0.4 -0.2 0.0 0.2 0.4

0 2 4 6 8

Density

gene normalized ER expression ER positive and negative samples

A

B

C

(13)

Supplementary Figure S8: Disease free survival of patients according to biochemical and microarray derived status for ER and PgR.

Kaplan Meier analysis of disease free survival of patients stratified according biochemical (A,D) or microarray (B,C,E,F) status of ER (A,B,C) and PgR (D,E,F). Either only samples with biochemical derived status (A,B,D,E) or all 2058 samples with follow up data (C,F) were included.

months

120 100 80 60 40 20 0

Disease free survival ^1.0

0.8

0.6

0.4

0.2

0.0

PgR by microarray (n=2058)

F

PgR⁺ (n=1204) PgR^- (n=854)

months

12 0 100 80 60 40 20 0

Disease free survival

PgR by microarray (n=1085)

E

1.0

0.8

0.6

0.4

0.2

0.0

PgR⁺ (n=612) PgR^- (n=473)

months

120 100 80 60 40 20 0

Disease free survival ^1.0

0.8

0.6

0.4

0.2

0.0

PgR biochemical (n=1085)

PgR⁺ (n=685) PgR^- (n=400)

D

months

120 100 80 60 40 20 0

1.0

0.8

0.6

0.4

0.2

0.0

ER by microarray (n=2058)

C

ER⁺ (n=1548) ER^- (n=510)

months

120 100 80 60 40 20 0

ER by microarray (n=1683)

B

1.0

0.8

0.6

0.4

0.2

0.0

ER⁺ (n=1252) ER^- (n=431)

months

120 100 80 60 40 20 0 1.0

0.8

0.6

0.4

0.2

0.0

ER biochemical (n=1683)

ER⁺ (n=1278) ER^- (n=405)

A

Disease free survival

(14)

Supplementary Figure S9: Disease free survival of untreated (A) and endocrine treated (B) patients stratified according to ER and PgR status based on microarray.

All patients were with available follow up information were selected which were either untreated or treated only with adjuvant endocrine therapy and which have both ER and PgR status available based on microarray as well as biochemical assay (n=722).

months

120 100

80 60

40 0 20

Disease free survival

Endocrine treatment (n=314)

B

1.0

0.8

0.6

0.4

0.2

0.0 ER

⁺

PgR

⁺

(n=220) ER

^-

PgR

^-

(n=17) ER

⁺

PgR

^-

(n=74) ER

^-

PgR

⁺

(n=3)

months

120 100

80 60

40 20

0 Disease free survival

1.0

0.8

0.6

0.4

0.2

0.0 ER

⁺

PgR

⁺

(n=198) ER

^-

PgR

^-

(n=87) ER

⁺

PgR

^-

(n=101) ER

^-

PgR

⁺

(n=22) Untreated (n=408)

A

(15)

Supplementary Figure S10: Comparison of Affymetrix expression values for ER, HER2 and PgR.

Distribution of expression values are given on an identical scale on the x‐axis (ProbeSets

205225_at, 216836_s_at, and 208305_at for ER, HER2 and PgR, respectively)

. Color codes represent the biochemical derived status of the samples for ER, HER2, and PgR.

PgR biochemical:

positive negative

PgR microarray

normalized log MAS5 values

‐0.02 ‐0.01 0 0.01 0.02

HER2 biochemical:

positive negative

HER2 microarray ER microarray

ER biochemical:

positive

negative

(16)

ER status:

PgR status:

HER2 status:

Supplementary Figure S11: Comparison of biochemical ER, HER2 and PgR status with microarray derived status using individual dataset specific cutoff values in a training‐validation approach.

Specific cutoff values derived from fitting in individual datasets (as training sets) were validated using all remaining samples with information on biochemical status (validation sets). The observed values for overall accuracy sensitivity and specificity are given for each cutoff derived from a single dataset.

0.0%

20.0%

40.0%

60.0%

80.0%

100.0%

NewYork Edinburgh EORTC Frankfurt Rotterdam MDA133 Frankfurt_2 San_Francisco Genentech Stockholm TransBIG Signapore expO Frankfurt_3 London Oxford_Untr… Veridex_Tam Mainz Uppsala Boston London_2 Oxford_Tam…

Accuracy Sensitivity Specificity

0.0%

20.0%

40.0%

60.0%

80.0%

100.0%

Frankfurt London_2 London Edinburgh Rotterdam MDA133 Uppsala Stockholm TransBIG Oxford_Tamo… Frankfurt_2 Mainz Frankfurt_3 expO EORTC NewYork Oxford_Untre… Veridex_Tam Signapore San_Francisco Boston Genentech

0.0%

20.0%

40.0%

60.0%

80.0%

100.0%

Edinburgh Mainz London Oxford_Tamoxi… San_Francisco Oxford_Untrea… TransBIG Uppsala Rotterdam Veridex_Tam Stockholm MDA133 London_2 expO Frankfurt_2 Genentech Frankfurt Signapore NewYork Boston Frankfurt_3 EORTC