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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‡