Download Supplemental Methods, Figures 1-2, Table S1

Klauschen et al.
Computer assisted Ki67 scoring in the GeparTrio breast cancer study cohort
Supplemental methods
Comparison of different intensity classes
Ki67 stained histological images are characterized by the presence of more than two intensity
classes, such as no tissue, tissue but no cell nuclei, Ki67 negative cell nuclei and Ki67 positive cell
nuclei each showing a certain haematoxylin intensity (Fig. 1A and B).
Supp. Figure 1: A) Ki67 stained breast cancer tissue. B) Haematoxylin Signal derived from color
deconvolution (1) and C) histogram showing frequencies (y axis) of specific heamatoxylin intensities (x
axis) of the image background (red) and foreground (green). Arrows show representative locations of
different intensity classes: no tissue (red), tissue but no cell nuclei (blue), Ki67 negative cell (green) and
Ki67 positive cell nuclei (orange).
Ki67 threshold finding method
The threshold finding method presented in this study is based on the assumption that the “real”
Ki67 staining fits the positive cell nuclei. That means that IHC staining signal that is found within
the image background or only partly matches the cell nuclei is considered a “false” (or
unspecific) signal. To compute the best matching Ki67 threshold first, the IHC- and
Hematoxylin counter-stain are separated through color deconvolution (1). Consecutively, all
possible thresholds ti (1..255) are tested and true positive (tp), false positive (fp) and false
negative (fn) events are computed as follows:

tp: number of Ki67 positive pixels that belong to a cell nucleus

fp: number of Ki67 positive pixels that belong to the image background

fn: number of Ki67 negative pixels that belong to a cell nucleus with at least one Ki67
positive pixel
Then the precision and recall measures are computed (Eq. 1 and 2) and outlined to the F-score
(Eq. 3) which is the harmonic mean of precision and recall.
𝑃𝑟𝑒𝑐𝑖𝑠𝑖𝑜𝑛 =
𝑡𝑝
𝑡𝑝 + 𝑓𝑝
(1)
Klauschen et al.
Computer assisted Ki67 scoring in the GeparTrio breast cancer study cohort
𝑅𝑒𝑐𝑎𝑙𝑙 =
𝐹 =2∗
𝑡𝑝
𝑡𝑝 + 𝑓𝑛
𝑃𝑟𝑒𝑐𝑖𝑠𝑖𝑜𝑛 ∗ 𝑅𝑒𝑐𝑎𝑙𝑙
𝑃𝑟𝑒𝑐𝑖𝑠𝑖𝑜𝑛 + 𝑅𝑒𝑐𝑎𝑙𝑙
(2)
(3)
In rare cases a moderate Ki67 “background” staining may occur that nearly perfectly fits the
cell nuclei, which results in a high F-score. Therefore, this (low intensity) signal would lead to
an incorrectly high number of Ki67 positive cells which is, however, not plausible. To avoid
using too low thresholds caused by this phenomenon we compute a plausibility measure,
which combines the threshold level and the resulting relative number of Ki67 positive cells.
Therefore, a border line is constructed were the relative threshold tRel (Eq. 4) and the resulting
ratio of positive cells are equal (Fig. 2) and where Imax is the maximum immune signal
occurring in the corresponding image.
𝑡𝑖
𝑡𝑟𝑒𝑙 = 𝑀𝑖𝑛(
, 1)
𝐼𝑚𝑎𝑥
(4)
All thresholds that result in a ratio of positive cells below this border line are considered
plausible (plausibility=1) or not plausible otherwise (plausibility=1-[Distance to borderline]).
Figure 2: Construction of the borderline between plausible and non-plausible thresholds by combining the
threshold and the resulting ratio of positive cells.
Finally, local maxima in the one-dimensional function F(i) are calculated and the
corresponding thresholds are used as potential thresholds. The threshold ti is selected where
the corresponding score (Eq. 5) is maximal.
Klauschen et al.
Computer assisted Ki67 scoring in the GeparTrio breast cancer study cohort
𝑆𝑐𝑜𝑟𝑒𝑖 = 𝐹(𝑖) ∗ 𝑃𝑙𝑎𝑢𝑠𝑖𝑏𝑖𝑙𝑖𝑡𝑦(𝑖)
(5)
Study population
In the neoadjuvant GeparTrio study (NCT00544765) patients with untreated unilateral
or bilateral primary breast cancer were enrolled after given written informed consent.
Eligibility required tumor diagnosis by core biopsy, plus at least one of the following risk
factors: age <36 years, clinical tumor size >5 cm, estrogen (ER) and progesterone (PR)
receptor negativity, clinical axillary node involvement, or tumor grade 3 (2-5). An
overview on the baseline clinicopathological parameters is given in (2) and Table S1.
Patients received between 2002 and 2005 six to eight cycles of docetaxel, doxorubicin
and cyclophosphamide (TAC) or a sequence of two cycles TAC followed by
vinorelbine/capecitabine depending on clinical response after two cycles. At the time of
the GeparTrio study there was not established central pathology for the GBG trials,
therefore the local data was used for hormone receptor status (in line with the study
protocol). For HER2 we have added data on some cases based on central testing, as
HER2 testing was not fully established at the time the study was conducted. No patient
received trastuzumab during neo- or adjuvant treatment. Postoperative radiotherapy
and endocrine treatment was given according to national guidelines. Ethic committee
approval was obtained for all centers participating in the clinical studies and from the
institutional review board of the Charité hospital.
Klauschen et al.
Computer assisted Ki67 scoring in the GeparTrio breast cancer study cohort
Supplementary table S1: Clinico-pathological data of the study cohort
No. of samples
Age group
< 40 years
≥ 40 years
Tumor type
Ductal/other
lobular
missing
Tumor grade
G1-G2
G3
missing
ER/PR Status (IHC)
ER-/PRER+ and/or PR+
HER2 status (IHC/FISH)
HER2 negative
HER2 positive
missing
Tumor type
Luminal/HER2Luminal/HER2+
HER2+/non-luminal
Triple-negative
missing
Clinical tumor stage
cT1-cT2
cT3-cT4
missing
Clinical nodal status
cN0
cN+
missing
Ki67
0-15%
15·1-35%
35·1-100%
Pathological response
no pCR
pCR
n
1082
%
100%
170
912
15.7
84.3
855
144
83
79
13.3
7.7
649
378
55
60
34.9
5.1
353
729
32.6
67.4
779
238
65
72
22
6
547
143
95
232
65
50.6
13.2
8.8
21.4
6
723
355
4
66.8
32.8
0.4
492
579
11
45.5
53.5
1
328
383
371
30.3
35.4
34.3
911
171
84.2
15.8
Klauschen et al.
Computer assisted Ki67 scoring in the GeparTrio breast cancer study cohort
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