SupplementaryTable 1.Basic characteristics of women in the study1,2,3

Participant Characteristics / Sulforaphane (n=27) / Placebo (n=27) / p
Mean (SD) / Mean (SD)
Age,yr / 53.52 (9.54) / 55.30 (14.27) / 0.59
BMI (kg/m2) at baseline / 26.69 (5.38) / 27.95 (5.80) / 0.42
Height (cm) at baseline / 165.8 (5.78) / 164.5 (5.88) / 0.42
Cruciferous vegetable intake (g/day) 5 / 83.0 (64.5) / 80.3 (50.3) / 0.86
n (%4) / n (%4)
Race
White
Non-White / 26 (96.3)
1 (3.7) / 24 (88.9)
3 (11.1) / 0.30
Family history of BreastCancer
Yes
No / 12 (44.4)
15 (55.6) / 8 (29.6)
19 (70.4) / 0.26
Smoking
Current
Former
Never
Missing / 0 (0.0)
7 (25.9)
17 (63.0)
3 (11.1) / 2 (7.4)
5 (18.5)
15 (55.5)
5(18.5) / 0.51
Alcohol
Current
Former
Never
Missing / 13 (48.1)
3 (11.1)
8 (29.6)
3(11.1) / 8(29.6)
3(11.1)
10 (37.0)
6 (22.2) / 0.55
Income
≤$25,000
$25,000-$50,000
>$50,000
Refuse/Don’t know/missing / 3(11.1)
2 (7.4)
14 (51.9)
8 (29.6) / 9 (33.3)
2 (7.4)
11 (40.7)
5 (18.5) / 0.20
Education
≤ 12 years
Some college/technical
≥ College graduate
Missing / 3 (11.1)
6 (22.2)
13 (48.1)
5(18.5) / 3 (11.1)
5 (18.5)
13 (48.1)
6(22.2) / 1.00
Marital status
Single, Divorced, Widowed
Married/partner
Missing / 10 (37.0)
14 (51.9)
3 (11.1) / 8 (29.6)
13 (48.1)
6 (22.2) / 0.81
Use of NSAIDS
Yes
No
Missing / 20 (74.1)
7 (25.9)
0 (0.0) / 8 (29.6)
18 (66.7)
1 (3.7) / 0.002*c
Menopausal Status6
Pre
Peri
Post / 8 (29.6)
8 (29.6)
11 (40.7) / 11 (40.7)
4 (14.8)
12 (44.4) / 0.40
Pathology Biopsy Diagnosis
Benign
Malignant7 / 15 (58.3)
12 (41.7) / 13 (50.0)
14 (50.0) / 0.21
1 t-tests were conducted for continuous variables (age, BMI,height and cruciferous vegetable intake);chi-square tests were conducted for categorical variables with expected cell frequencies ≥ 5; and Fisher’s exact tests were conducted for categorical variables with expected cell frequencies < 5.
2 p-value for t-tests or chi-square tests between supplement and placebo groups, *p <0.05.
3For the categorical variables with missing values, chi-square tests or Fisher’s exact tests were conducted without including missing group.
4 Percentages may not add up to 100 due to rounding values.
5Cruciferous vegetable intake includes sources from food lines, mixed dishes and condiments.
6 For the 13 women that did not report their menopausal status, those aged45 years were grouped into Pre-menopausal status, those aged between 45 and 55 years were grouped into Peri-menopausal status, and those aged > 55 years were grouped into Post-menopausal status. The chi-square testp-value between supplemental and placebo groupsamong the 41 women who initially reported menopausal status was 0.86.
7In supplement group, malignantcategory includes 7 subjects having DCIS (ductal carcinoma in situ) or DCIS+atypical ductal hyperplasia (ADH) and 5 subjects having invasive cancer with DCIS component; in placebo group, malignantcategory includes 10 subjects having DCIS/DCIS+ADH and 4 subjects having invasive cancer with DCIS component.

SupplementaryTable 2. Incidence of reported grade 2 adverse events in the BroccoMax™ trial (treatment-related)

Adverse Events (AE) / Sulforaphane (n=27) / Placebo (n=27) / p1
Number / %2 / Number / %2
Bloating / 5 / 18.5 / 5 / 18.5 / 1.00
Gas/Flatulence / 1 / 3.7 / 4 / 14.8 / 0.35
Burping / 0 / 0 / 0 / 0 / 1.00
Diarrhea / 1 / 3.7 / 2 / 7.4 / 1.00
Nausea/vomiting / 0 / 0 / 1 / 3.7 / 1.00
Headache / 1 / 3.7 / 3 / 11.1 / 0.61
Taste Alteration / 0 / 0 / 2 / 7.4 / 0.49
Bruising / 1 / 3.7 / 1 / 3.7 / 1.00
Other3 / 5 / 18.5 / 7 / 25.9 / 0.74
All4 / 8 / 29.6 / 9 / 33.3 / 1.00

1 Fisher’s exact tests.

2 Number of subjects reporting event/total number of study subjects (%).

3 Other changes to health included tingling tongue sensation, tiredness, change ofsleep pattern, arthritic pain, allergy, heartburn, bulbitis, cramping.

4 Count of subjects who experienced at least one of the grade 2 adverse events.

SupplementaryTable 3. Sulforaphane (SFN) and SFN metabolite levels in urine and plasma as well as histone deacetylase (HDAC) activity changes from pre- to post-treatment by treatment group

Sulforaphane
Mean (SE) / Placebo
Mean (SE) / p3
Urinary Metabolites
Pre-intervention / Post-intervention / Changes2 / Pre-intervention / Post-intervention / Changes2
n =21 / n =22 / n =21 / n =22 / n =22 / n =22
Urinary Creatinine / 13.0 (1.80) / 10.83 (1.61) / -1.75 (2.52) / 11.07 (1.46) / 9.56 (1.53) / -1.50 (1.33) / 0.63
Total SFN Metabolites1 / 0.07 (0.01) / 1.06 (0.32) / 1.00 (0.33) / 0.10 (0.02) / 0.05 (0.01) / -0.05 (0.02) / <0.0001
SFN-NAC / 0.02 (0.01) / 0.77 (0.24) / 0.75 (0.25) / 0.04 (0.01) / 0.02 (0.00) / -0.02 (0.01) / <0.0001
SFN-Cys / 0.02 (0.01) / 0.22 (0.06) / 0.20 (0.06) / 0.04 (0.01) / 0.02 (0.00) / -0.02 (0.00) / <0.0001
SFN-GSH / 0.00 (0.00) / 0.01 (0.00) / 0.00 (0.01) / 0.01 (0.00) / 0.00 (0.00) / -0.01 (0.00) / 0.0004
SFN-CG / 0.01 (0.00) / 0.02 (0.01) / 0.01 (0.01) / 0.02 (0.00) / 0.01 (0.00) / -0.01 (0.00) / 0.03
SFN / 0.00 (0.00) / 0.05 (0.02) / 0.05 (0.02) / 0.00 (0.00) / 0.00 (0.00) / 0.00 (0.00) / <0.0001
Plasma Metabolites
Pre-intervention / Post-intervention / Changes2 / Pre-intervention / Post-intervention / Changes2
n =16 / n =17 / n =16 / n =18 / n =21 / n =18
Total SFN Metabolites1 / 0.00 (0.00) / 0.25 (0.07) / 0.27 (0.07) / 0.00 (0.00) / 0.00 (0.00) / 0.00 (0.00) / <0.0001
SFN-NAC / 0.00 (0.00) / 0.07 (0.01) / 0.08 (0.01) / 0.00 (0.00) / 0.00 (0.00) / 0.00 (0.00) / <0.0001
SFN-Cys / 0.00 (0.00) / 0.02 (0.02) / 0.03 (0.03) / 0.00 (0.00) / 0.00 (0.00) / 0.00 (0.00) / 0.32
SFN-GSH / 0.00 (0.00) / 0.06 (0.01) / 0.07 (0.01) / 0.00 (0.00) / 0.00 (0.00) / 0.00 (0.00) / <0.0001
SFN-CG / 0.00 (0.00) / 0.10 (0.04) / 0.10 (0.05) / 0.00 (0.00) / 0.00 (0.00) / 0.00 (0.00) / 0.03
HDAC Activity in peripheral blood mononuclear cells (PBMC)
Pre-intervention / Post-intervention / Changes2 / Pre-intervention / Post-intervention / Changes2
n =23 / n =24 / n =23 / n =25 / n =24 / n =24
HDAC Activity4 / 447.51 (48.80) / 371.14 (44.78) / -80.39 (48.53) / 432.76 (42.00) / 476.57 (43.83) / 27.52 (32.58) / 0.04
1 Urinary SFN metabolite concentration units = micromolar(µM) concentrationsof urinary SFN metabolite / millimolar (mM) concentrations of urinary creatinine.Plasma SFN metabolite concentrations are shown in micromolar(µM) concentrations.
2 Change = post-intervention level minus pre-intervention level. The mean changes value may not be equal to mean in the post-intervention group minus mean in the pre-intervention group due to different sample sizes in these two groups.
3p-values were calculated for difference of change in means across treatment groups. t-test was used for change of HDAC activity, which had normal distribution; Mann-Whitney U test was used for all other variables due to non-normal distribution.
4 HDAC activity values represent pmol/min/mg protein.

SupplementaryTable 4. Log2-transformed LSMEANS of immunohistochemistry H-score of selected breast tissue biomarkers in women scheduled for breast biopsy participating the BroccoMax™ trial

n of Subjects / n of Observations Used2 / SFN Group / Placebo Group / p
comparing treatment groups1 / False Discovery Rate p
Values5
Pre-to-Post Change
LSMEANS(95% CI)3 / Pre-to-Post Change
LSMEANS(95% CI)3
H3K18
Benign / 28/31 / -1.15 (-3.60, 1.29) / -0.57 (-3.90, 2.76) / 0.32 / 0.70
DCIS / 15/22 / -1.32 (-2.91, 0.26)a / -0.55 (-1.65, 0.55) / 0.35 / 0.70
IDC / 13/20 / -0.30 (-2.92, 2.32) / -0.29 (-3.18, 2.60) / 0.99 / 0.99
H3K9
Benign / 38/48 / -1.10 (-2.39, 0.19) a / -1.42 (-2.60, -0.25)b / 0.68 / 0.79
DCIS / 16/27 / -0.78 (-2.56, 1.00) / -1.38 (-2.49, -0.28)b / 0.53 / 0.73
IDC / 14/17 / -0.02 (-6.08, 6.03) / -1.81 (-6.22, 2.60) / 0.40 / 0.70
HDAC34
Benign / 31/35 / -1.29 (-2.70, 0.12)b / -0.38 (-1.80, 1.03) / 0.19 / 0.70
DCIS / 9/11 / n/a / n/a / n/a / n/a
IDC / 3/4 / n/a / n/a / n/a / n/a
HDAC64
Benign / 31/38 / -0.26 (-1.59, 1.07) / -0.70 (-2.04, 0.64) / 0.57 / 0.73
DCIS / 12/13 / n/a / n/a / n/a / n/a
IDC / 6/6 / -0.26 (-17.48, 17.99) / n/a / n/a / n/a
Ki-67
Benign / 46/60 / -1.40 (-2.29, -0.52)c / 0.21 (-0.70, 1.12) / 0.01 / 0.14
DCIS / 18/27 / 0.42 (-1.19, 2.03) / -0.60 (-1.88, 0.68) / 0.28 / 0.70
IDC / 12/15 / 1.05 (-3.66, 5.77) / 0.25 (-3.18, 3.67) / 0.32 / 0.70
p21
Benign / 45/61 / -0.72 (-1.78, 0.34) / -0.25 (-1.16, 0.66) / 0.47 / 0.73
DCIS / 19/28 / -0.57 (-2.43, 1.30) / -0.77 (-2.33, 0.79) / 0.85 / 0.92
IDC / 12/17 / 0.25 (-2.39, 2.88) / -1.71 (-4.87, 1.46) / 0.21 / 0.70
1 Mixed effect model adjusting NSAIDs useand length of intervention.
2 Each subject has maximum of 2 observations.
3 The unit of the LSMEANS is log2(H-score).
4 HDAC3 and HDAC6 were added late in the study. Differing numbers of participant specimens available reflect reasons such as: depleted blocks, tissue falling off stained slides and non-existent epithelial tissue to stain/review.
5 Multiple comparison adjusted p-values using the Benjamini–Hochberg False Discovery Rate. a p-value ≤ 0.1
b p-value ≤ 0.05 c p-value ≤ 0.01.