Appendix

Supplementary data (online only) to:Marleen Kok, Wilbert Zwart, Caroline Holm, Renske Fles, Michael Hauptmann, Laura J. Van ’t Veer, Lodewyk F.A. Wessels,Jacques Neefjes, Olle Stål, Sabine C. Linn, Göran Landberg, Rob MichalidesPKA-induced phosphorylation of ERα at serine 305 and/or high PAK1 levels predict tamoxifen resistance in the majority ofER positive breast cancer cases

Figure A1. Staining for the catalytic subunit of PKA phosphorylated at threonine 197.

A) Representative example of positive pPKA staining. B) Absence of pPKA expression after dephosphorylation via incubation with lambda phosphatase on the tumor slide prior to immunohistochemistry.

A B

564 randomized patientsStart of trial

70 excluded (no FFPE tissue and no data on ERα)

494 with ERα data

384 ERα >10%

110 excluded (ERα ≤10%)

231 with ERα305-P, pPKA or PAK1 data

153 excluded (no ERα305-P, pPKA and PAK1 data)

262 events

32 events

230 events

55 events

175 events

71 events

104 events

Figure A2. Flow diagram of patients

Flow of patients through the study including number of patients in each stage. Reasons for dropout and number of events (recurrence) in each subgroup are listed.

N=231Log-rank=0.020

HR=0.63, 95% CI 0.42-0.93, p=0.021

N=384Log-rank=0.003

HR=0.64, 95% CI 0.47-0.86, p=0.004

Figure A3. Overall tamoxifen benefit in patients included in the current translational study (n=231) compared to tamoxifen benefit in all ER-positive breast cancer patients included in the trial (n=384).

Kaplan-Meier analysis of recurrence-free survival according to randomization in A) 384 patients of whom tumor material was available and the ER was expressed in >10% of tumor cells and in B) 231 patients of whom tumor material was available, the ER was expressed in >10% of tumor cells and data on PAK1, PKA and ER305-P were available.

---- tamoxifen, --- no adjuvant systemic treatment

NKI-AVL (training series)

Protein Supplier Catalogusnr Dilution Pretreatment Scoring Reference

ERα Labvision/Neomarkers MS-750-S 50x citrate nuclear, percentage of cells Hannemann et al.

PR Immunologic ACD 14 400x citrate nuclear, percentage of cells Kok et al.

HER2 Thermo Scientific RM-9103-S 25x citrate membranous, 0/1/2/3 Van de Vijver et al.

PAK1 Cell Signaling 2602.00 25x citrate intensity 0-5, nuclear 0/1 Holm et al.

Phospho-ERα-S305 Millipore 124-9-4 20x citrate nuclear, percentage of cells Holm et al.

Phospho-PKA Cell Signaling 4781 40x citrate intensity 0-5, nuclear 0/1 x

Lund (validation series)

Protein Supplier Catalogusnr Dilution Pretreatment Scoring Reference

ERα Ventana Medical Systems clone 6F11 prediluted citrate nuclear, percentage of cells Ryden et al.

Ki-67 Dako M7240 200x citrate nuclear, percentage of cells Jirstrom et al.

PAK1 Cell Signaling 2602.00 25x citrate intensity 0-5, nuclear 0/1 Holm et al.

Phospho-ERα-S305 Millipore 124-9-4 20x citrate nuclear, percentage of cells Holm et al.

Phospho-PKA Cell Signaling 4781 40x citrate intensity 0-5, nuclear 0/1 x

Table A1. Details immunohistochemistry

Table A2. Distribution of pPKA according to clinicopathological parameters (training series NKI)

pn (%) n (%)

Total 30 (29) 73 (71)Median age at surgery 59 60 0.61 *Grade I/II 21 (70) 50 (68) 1.00

III 9 (30) 23 (32)

LN status 0 25 (83) 43 (61) 0.036

(2 unknown) >0 5 (17) 28 (39)

WHO subtype IDC 23 (82) 64 (90) 0.31

(4 unknown or other subtype) ILC 5 (18) 7 (10)

Size <=20 mm 17 (57) 44 (60) 0.56> 20 mm 13 (43) 29 (40)

ERα (IHC) 11-79% 10 (33) 17 (23) 0.33>= 80% 20 (67) 56 (77)

PR (IHC) <= 10% 14 (47) 22 (30) 0.120> 10% 16 (53) 51 (70)

HER2 (IHC) 0,1,2 26 (87) 63 (86) 1.003 4 (13) 10 (14)

PKA scoring is based on expression in the cytoplasm

Percentages might not add up to 100% because of rounding

P-value are based on Fisher's Exact tests (* Mann-Whitney U test)

Abbreviations:

pPKA= phosphorylation of protein kinase A at threonine 197

Grade= histological grading based on Nottingham Grading system

LN= lymph node

IDC= invasive ductal carcinoma

ILC= invasive lobular carcinoma

ERα= oestrogen receptor α

PR=progesterone receptor

HER2= human epidermal growth factor 2

IHC= immunohistochemistry

pPKA-

negative

pPKA-

positive

Insufficient data for Score Data for Score available

Variable Category n % n % P-value

Total 153 231

Age at surgery Median in years (range) 45 (27-57) 45 (26-57) 0.25¶

Grade* I/IIIIIUnknown

1013913

72%28%

151773

66%34%

0.25

Lymph node status NegativePositive

39114

25%75%

55176

24%76%

0.72

WHO subtype Invasive Ductal CarcinomaInvasive Lobular CarcinomaUnknown or other type

1041435

88%12%

1962312

89%11%

0.72

Size ≤ 20 mm> 20 mmUnknown

64881

42%58%

97134

42%58%

1.00

Progesterone receptor (IHC) ≤ 10%> 10%Unknown

119448

10%90%

2219613

10%90%

1.00

Ki67 (IHC) ≤ 25%> 25%Unknown

861453

86%14%

1653828

81%19%

0.34

Table A3 . Randomized controlled trial (Lund) used as validation series. Distribution of clinico-pathological parameters in the excluded cases (ERα>10%, (no data to generate PAK1-PKA/ ERS305-P Score, n=153) vs included patients in the validation series (n=231).

* According Nottingham Grading system (Elston et al.).

Table A4. Distribution of prognostic factors in the subgroup of tumors classified as sensitive by the PAK1-PKA/ ERS305-P Score in the validation set (PAK1 and/or PKA/ER305-P negative) according to systemic treatment received (tamoxifen versus no adjuvant systemic treatment).

* According Nottingham Grading system (Elston et al.).† Mann-Whitney U test

‡ Fisher’s Exact test

Abbreviations: LN lymph node status, ER estrogen receptor, PR progesterone receptor, IDC invasive ductal carcinoma, ILC invasive lobular carcinoma.

Tamoxifen Control P-value

Variable Category N=78 N=91

Age at surgery Median(range) 45 (36-57) 46 (26-52) 0.79†

LN NegativePositive

24 (31%)54 (69%)

18 (20%)73 (80%)

0.11‡

Size <= 20 mm> 20 mm

28 (36%)50 (64%)

38 (42%)53 (58%)

0.53‡

Grade* I/IIIIIUnknown

48 (62%)29 (38%)1

61 (68%)29 (32%)1

0.52‡

ER levels >10,<75%>75%Unknown

30 (39%)47 (61%)1

36 (41%)52 (59%)3

0.87‡

PR <=10%>10%Unknown

9 (13%)62 (87%)7

9 (10%)77 (90%)5

0.80‡

WHO IDCILCUnknown/other

66 (89%)8 (11%)4

73 (86%)12 (14%)6

0.63‡