Supplementary Information

DNA Methylation Markers for Breast Cancer Detection in the

Developing World: Marker Training, Assay Validation, Automation and

Pilot Testing

Bradley M. Downs, Claudia Mercado-Rodriguez, Ashley Cimino-Mathews, Chuang Chen, Jing-

Ping Yuan, Eunice van den Berg, Leslie M. Cope, Fernando Schmitt, Gary Tse, Syed Z. Ali,

Danielle Meir-Levi, Rupali Sood, Juanjuan Li, Andrea Richardson, Suzana Tulac, Kriszten J.

Kocmond, Timothy de Guzman, Edwin W. Lai, Brian Rhees, Michael Bates, Antonio C. Wolff,

Susan C. Harvey, Monica Rizzo, Marina B. Mosunjac, Edward Gabrielson, Christopher B.

Umbricht, Kala Visvanathan, Mary Jo Fackler, Saraswati Sukumar

Supplementary Tables 1-3 pages 2-5

Supplementary Figures, 1-6 pages 6-11

1

Supplementary Tables

Supplementary Table S1

Individual Gene Marker Selection (25 markers)AKR1B1 APC CCND2 COL6A2 HIST1H3C HOXB4 RASGRF2 RASSF1 TMEFF2 ZNF671

Tissue Ca. B Ca. B Ca. B Ca. B Ca. B Ca. B Ca. B Ca. B Ca. B Ca. BNumber of Samples 107 114 109 116 103 110 109 116 107 116 107 113 102 112 108 114 103 113 107 112% M per sample:Minimum 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 025th Percentile 0 0 0 0 0 1 0 0 0 0 0 0 1 0 0 0 0 0 0 075th Percentile 10 0 33 0 10 1 6 1 19 0 5 0 26 2 27 2 11 0 8 1Maximum 62 4 90 24 74 14 82 22 82 3 79 0 85 12 88 26 99 15 75 19

ARHGEF7 CBF2T3 CDKL2 cg18191418 EVI1 GAS7 GPX7 HIN MAL NEK9Tissue Ca. B Ca. B Ca. B Ca. B Ca. B Ca. B Ca. B Ca. B Ca. B Ca. BNumber of Samples 108 115 22 11 49 63 22 10 21 25 7 5 70 72 64 53 96 107 24 12% M per sample:Minimum 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 025th Percentile 0 0 0 0 8 2 0 0 1 3 0 0 0 0 0 0 0 0 0 075th Percentile 0 0 0 0 53 8 0 0 19 6 2 0 5 2 4 0 7 0 0 0Maximum 84 5 8 0 68 44 6 0 86 24 3 0 94 20 37 69 69 5 0 0

RARB TM6SF1 TREX1 TWIST1 WNT1Tissue Ca. B Ca. B Ca. B Ca. B Ca. BNumber of Samples 106 113 67 74 32 13 74 75 32 13% M per sample:Minimum 0 0 0 0 0 0 0 0 0 025th Percentile 1 0 0 4 0 0 0 0 0 075th Percentile 4 2 18 10 0 1 7 1 0 0Maximum 98 7 84 35 10 4 42 4 23 0Ca. = Cancer (IDC, DCIS), B = Benign/Normal; % M = Percent methylation of gene. The final 10 gene marker set is shown in the top row. This table accompanies Supplementary Figure S1.

2

Supplementary Table S1. Selection of marker gene-panel in the Training cohort. Percent methylation (minimum, 25th and 75th percentile, and maximum) is shown for each of 25 genes analyzed by QM-MSP in the training cohort of FFPE breast cancer and benign/normal samples. Sixteen markers showed considerably higher median methylation in cancer than in benign/normal (P<0.01, based on the Mann-Whitney test). To further minimize the risk of false positives, each remaining marker was examined to determine that levels of methylation in benign and normal samples were low. Specifically, the 75 th percentile of methylation was calculated separately in tumor and benign samples, discarding any markers in which the 75th percentile of normal methylation was high, or where the difference between normal and tumor was small (also cross-reference Figure S1). The ten genes that were finally selected are denoted in red. Ca. cancer; B, benign; ns, not significant. This table accompanies Supplementary Figure S1.

3

Mann Whitney Test P-Value *

ER

/PR

+, H

ER

2-

ER

/PR

+, H

ER

2+

ER

/PR

-, H

ER

2+

ER

/PR

-, H

ER

2-

Mann Whitney Test P-Value *

ER

/PR

+, H

ER

2-

ER

/PR

+, H

ER

2+

ER

/PR

-, H

ER

2+

ER

/PR

-, H

ER

2-

AKR1B1 HOXB4ER/PR+, HER2- ER/PR+, HER2-ER/PR+, HER2+ 0.790 ER/PR+, HER2+ 0.288ER/PR-, HER2+ 0.201 0.092 ER/PR-, HER2+ 0.962 0.332ER/PR-, HER2- 0.002 0.009 0.004 ER/PR-, HER2- 0.085 0.795 0.164

APC RASGRF2ER/PR+, HER2- ER/PR+, HER2-ER/PR+, HER2+ 0.865 ER/PR+, HER2+ 0.731ER/PR-, HER2+ 0.035 0.054 ER/PR-, HER2+ 0.611 0.545ER/PR-, HER2- 0.053 0.170 0.002 ER/PR-, HER2- 0.020 0.145 0.146

CCND2 RASSF1ER/PR+, HER2- ER/PR+, HER2-ER/PR+, HER2+ 0.390 ER/PR+, HER2+ 0.717ER/PR-, HER2+ 0.387 0.940 ER/PR-, HER2+ 0.543 0.283ER/PR-, HER2- 0.001 0.155 0.149 ER/PR-, HER2- <0.0001 0.007 0.002

COL6A2 TMEFF2ER/PR+, HER2- ER/PR+, HER2-ER/PR+, HER2+ 0.310 ER/PR+, HER2+ 0.969ER/PR-, HER2+ 0.033 0.295 ER/PR-, HER2+ 0.009 0.077ER/PR-, HER2- 0.079 0.017 0.003 ER/PR-, HER2- 0.145 0.333 0.001

HIST1H3C ZNF671ER/PR+, HER2- ER/PR+, HER2-ER/PR+, HER2+ 0.824 ER/PR+, HER2+ 0.251ER/PR-, HER2+ 0.695 0.820 ER/PR-, HER2+ 0.099 0.562ER/PR-, HER2- 0.057 0.216 0.424 ER/PR-, HER2- <0.0001 0.059 0.420

Comparison Between IHC Subtypes for Methylation Levels of Individual Markers

Supplementary Table S2B

* Not adjusted for the false discovery rate (FDR)

This table accompanies Supplementary Figure S5A.

Supplementary Table S3

Incidence of Methylation by Region using the 10-gene panel

Diagnosis Location Methylated* Not Methylated

% Methylated P**

Cancer (IDC+ILC+DCIS)

U.S. 81 11 88

0.66China 64 10 86

S. Africa 48 4 92

IDC

U.S. 41 4 91

0.78China 53 6 90

S. Africa 25 1 96

ILC

U.S. 21 2 91

NAChina - - -

S. Africa - - -

DCIS

U.S. 19 5 79

0.47China 11 4 73

S. Africa 23 3 88

* Above the threshold of 14.5 cumulative methylation units

** Fisher Exact for 3-way comparison between regions

This table accompanies Supplementary Figure S5B, C.

4

Supplementary Table S4

ROC Sensitivity and Specificity Over a Range of Thresholds for the Cartridge Assay

Threshold Sensitivity 95% CI Specificity 95% CILikelihood

Ratio

> 8.2 95.8 78.9% to 99.9% 75.5 61.1% to 86.7% 3.91

> 9.55 95.8 78.9% to 99.9% 77.6 63.4% to 88.2% 4.27

> 9.78 95.8 78.9% to 99.9% 79.6 65.7% to 89.8% 4.7

> 11.2 95.8 78.9% to 99.9% 81.6 68% to 91.2% 5.22

> 12.9 95.8 78.9% to 99.9% 83.7 70.3% to 92.7% 5.87

> 13.7 95.8 78.9% to 99.9% 85.7 72.8% to 94.1% 6.71

> 14.2 95.8 78.9% to 99.9% 87.8 75.2% to 95.4% 7.83

> 18.3 95.8 78.9% to 99.9% 89.8 77.8% to 96.6% 9.39

> 22.5 91.7 73% to 99% 89.8 77.8% to 96.6% 8.98

> 23.3 91.7 73% to 99% 91.8 80.4% to 97.7% 11.2

> 24 91.7 73% to 99% 93.9 83.1% to 98.7% 15.0

> 25 91.7 73% to 99% 95.9 86% to 99.5% 22.5

> 30.1 91.7 73% to 99% 98 89.1% to 99.9% 44.9

> 35.5 87.5 67.6% to 97.3% 98 89.1% to 99.9% 42.9

> 36.9 83.3 62.6% to 95.3% 98 89.1% to 99.9% 40.8

This table accompanies Figure 5.

ROC, receiver operator characteristic. Sensitivity and specificity with 95% CI using the threshold used in Figure 5, derived in the Test set using QM-MSP analysis of FFPE tissues, is underlined.

5

IDCDCIS

BENIGN NORMAL

0

20

40

60

80

100 AKR1B1

% M

ethy

latio

n

P < 0.0001

N = 107 114

IDCDCIS

BENIGN NORMAL

APC

P <0.0001

109 116

IDCDCIS

BENIGN NORMAL

ARHGEF7

P <0.0001

108 115

IDCDCIS

BENIGN NORMAL

CBF2T3

22 11

BENIGN NORMAL

IDCDCIS

CCND2

P = 0.0004

103 110

IDCDCIS

BENIGN NORMAL

CDKL2

P <0.0001

49 63

IDCDCIS

BENIGN NORMAL

0

20

40

60

80

100 EVI1

% M

ethy

latio

n

N = 21 25

IDCDCIS

BENIGNNORMAL

cg18191418

22 10

IDCDCIS

BENIGN NORMAL

GPX7

70 72

IDCDCIS

BENIGN NORMAL

HIN1

P =0.0063

64 53

IDCDCIS

BENIGN NORMAL

GAS7

7 5

IDCDCIS

BENIGN NORMAL

HIST1H3C

P <0.0001

107 116

IDCDCIS

BENIGN NORMAL

HOXB4

P <0.0001

107 113

IDCDCIS

BENIGN NORMAL

MAL

P <0.0001

96 107

IDC/DCIS

BENIGN/ NORMAL

NEK9

24 12

IDCDCIS

BENIGN NORMAL

RARB

P <0.0001

106 113

IDCDCIS

BENIGN NORMAL

RASGRF2

P <0.0001

102 112

IDCDCIS

BENIGN NORMAL

0

20

40

60

80

100 RASSF1

%M

ethy

latio

n

P <0.0001

N = 108 114

IDCDCIS

BENIGN NORMAL

0

20

40

60

80

100 TM6SF1

% M

ethy

latio

n

69 76N=

IDCDCIS

BENIGN NORMAL

TMEFF2

P <0.0001

103 113

IDCDCIS

BENIGN NORMAL

TREX1

32 13

IDCDCIS

BENIGN NORMAL

TWIST1

P =0.0006

74 75

IDCDCIS

BENIGN NORMAL

WNT1

32 13

IDCDCIS

BENIGN NORMAL

ZNF671

P =0.001

107 112

IDCDCIS

BENIGN NORMAL

COL6A2

P <0.0001

109 116

Supplementary Figure S1. Evaluation of the 25 Candidate Gene Markers. The percent (%) methylation for 25 individual breast cancer-associated genes was determined using the QM-MSP assay in the training cohort. Box plots depict gene methylation intensity (Y-axis, % methylation) in cancer [invasive ductal carcinomas (IDC) and ductal carcinoma in situ (DCIS)] versus

6

Supplementary Figure 1

Benign/Normal samples (X-axis). Significant Mann-Whitney P-values (< 0.01) are shown. N= number of samples in each group.

Supplementary Figure 2

Diagnosis Sensitivity (95% CI) Specificity (95% CI) AUC (95% CI)IDC 91% (81-97%) 88% (80-94%) 0.960 (0.925-0.989)ILC 91% (72-99%) 88% (80-94%) 0.998 (0.841-0.998)

DCIS 77% (58-90%) 88% (80-94%) 0.881 (0.777-0.962)

7

Supplementary Figure S2. Comparison Of Performance of the 10-gene Panel in Invasive Cancer versus DCIS in the Test Set.

Performance of the 10-gene panel (as measured by area under the ROC curve) assessed separately in invasive carcinomas (IDC, ILC) and pre-invasive ductal carcinoma in situ (DCIS). The dashed line indicates our target sensitivity at 90%, and dots indicate the cutoff at 14.5 CMI units on each curve. Sensitivity, specificity and AUC values with 95% CI are shown in the table. This figure accompanies Figure 3 in the main text.

Supplementary Figure S3

A.

B. C.

Age-associated risk of False Positives Logistic Regression Model of Benign/Normal

Coeff. Std. Err z-stat p-valueIntercept -4.043 1.037 -3.901 <0.00000Age 0.038 0.020 1.931 0.0535

Age-associated risk of False Negatives Logistic Regression Model of Tumor

Coeff. Std. Err z-stat p-valueIntercept 1.258 0.931 1.350 0.177Age 0.012 0.017 0.709 0.478

Linear Regression Model of CMI by Age response=log2(CMI), R2= 0.017

Coeff. Std. Err t-stat p-value

Intercept 2.187 0.293 7.453 <0.00000Age 0.011 0.006 1.866 0.0634

8

Supplementary Figure S3. Effect of Patient Age on Methylation. A. A linear regression model of CMI as a function of age, fit on benign/normal samples (Panel A) demonstrates a modest increase in methylation with greater age. Benign/Normal samples are shown in red, tumors in blue. The fitted model is shown as a solid red line, and the threshold of 14.5 CMI units is represented as a dashed line. CMI was log transformed to provide symmetry to the distribution (base 2, calculated after adding 1 to each CMI value to accommodate zeros in the data). The coefficient of 0.011 for age corresponds with a doubling of CMI from 5 to 10 units over 91 years.

B. Logistic regression models of misclassification rates as a function of age, fit on benign/normal samples shows a modest increase in the log-odds of false positives (samples as dots on top X-axis) with increased age (P = 0.0535).

C. The corresponding model for tumor samples shows a decrease in the risk of false negatives (samples as dots on bottom X-axis) for older patients, although the effect is very small and is not statistically significant (P = 0.478).

Linear Regression Model of CMI by Sample Age response=log2(CMI), R2= 0.017

Coeff. Std. Err t-stat p-valueIntercept 2.856 0.101 28.403 <0.00000Age -0.031 0.017 1.863 0.0639

9

Supplementary Figure S4

Supplementary Figure S4. Effect of Sample Age on Methylation.

Linear regression model of CMI as a function specimen (block) age, fit on benign/normal samples, shows a modest decrease in methylation with greater block age. Benign/Normal samples are shown in red, tumors in blue. The fitted model is shown as a solid red line, and the 14.5 CMI threshold is represented as a dashed line. CMI was log transformed to provide symmetry to the distribution (base 2, calculated after adding 1 to each CMI value to accommodate zeros in the data).

A. Methylation by Histologic Subtype

% Methylation in Cancer of Individual GenesCumulative Methylation of the 10-marker Panel ER/PR+, HER2- ER/PR+, HER2+ ER/PR-, HER2+ ER/PR-, HER2-

U.S. China S. Africa

% Methylation in Cancer of Individual GenesCumulative Methylation of 10-marker Panel

B. Methylation By Region

0

20

40

60

80

100

AKR1B10.004

APC0.020

CCND20.011

COL6A20.014

HIST1H3C0.283

HOXB40.265

RASGRF20.116

RASSF10.001

TMEFF20.019

ZNF6710.001

% M

ethy

latio

nP =ER/PR+,

HER2-ER/PR+,HER2+

ER/PR-, HER2+

ER/PR-, HER2-

0

100

200

300

400

63 14 22N =

CM

I

P = 0.077* ER/PR+, HER2-ER/PR+, HER2+ER/PR-, HER2+

* Kruskal-Wallis Test

9

ER/PR-, HER2-

0

20

40

60

80

100

AKR1B10.874

APC0.688

CCND20.010

COL6A20.003

HIST1H3C0.003

HOXB40.588

RASGRF20.705

RASSF10.705

TMEFF20.866

ZNF6710.043

% M

ethy

latio

n

P =

0

100

200

300

400

IDC/ILCDCIS

BenignNormal

92 74 52 61 101 46

CM

I

N =

P = 0.474*

P = 0.265*

* Kruskal-Wallis Test

C. Methylation AUCs by Region

Region Sensitivity (95% CI) Specificity (95% CI) AUC (95% CI)U.S. 88% (76-95%) 79% (60-92%) 0.941 (0.890-0.983)

China 81% (65-92%) 90% (78-97%) 0.901 (0.813-0.970)S. Africa 96% (80-100%) 96% (77-100%) 0.958 (0.872-1.000)

10

Supplementary Figure S5. Evaluation of Differences in Gene Methylation by Country of Origin and Immunohistochemical (IHC) Subtype. A. Methylation by IHC Subtype. DNA methylation in ER/PR+, HER2- (red), ER/PR+, HER2+ (blue), ER/PR,HER+ (fushcia), ER/PR-, HER2- (green). See Supplementary Tables S2A, S2B. B. Methylation by Region. DNA methylation in breast cancer from the U.S. (red), China (blue), and S. Africa (green). For A) and B), box plots indicate the levels of cumulative methylation (CMI) of the 10-gene marker panel and scatter plots indicate % methylation of individual genes as assessed by QM-MSP. For multiple comparison between groups the Kruskal-Wallis test, unadjusted P-values are indicated (*). N = number of samples. C. Performance of the 10-gene panel (as measured by area under the ROC curve) assessed separately in tissues from the U.S, China and Africa. The dashed line indicates our target sensitivity at 90%, and dots indicate the cutoff at 14.5 CMI units on each curve. Sensitivity, specificity and AUC values with 95% CI are shown in the table.

Supplementary Figure S5

Marker Set 1

0

5

10

15CCND2

AKR1B1

TMEFF2APC

ZNF671

100502512.56.253.12% Methylation

Ct (

Gen

e - A

CTB

)

Marker Set 2

-5

0

5

10COL6A2

HIST1H3CRASGRF2HOXB4RASSF1

100502512.56.253.12% Methylation

C

t (G

ene

- AC

TB)

12

11

Supplementary Figure S6. The Automated Cartridge Assay displays a Broad Dynamic Range for Detection of DNA Methylation. DNA aliquots prepared from CAMA-1 cell line (100% methylated for 10 targets) and human sperm (0% methylated) were independently adjusted to a dilution at which 1 μl DNA = Ct 21 (cycle threshold) when measured in the cartridge assay. DNAs were then mixed to make a dilution curve ranging from 100% - 3.12% methylation. Plotted is the average ∆Ct (Ct Gene - Ct ACTB; linear Y-axis) at each percentage of methylation (log2 X-axis), using nonlinear fit regression and 6 replicates per point for each of Gene Set 1 and Set 2, as indicated.

Supplementary Figure S6