Download Supplemental Fig. 4 (continued)

Supplemental Figures
Loss of circadian clock gene expression is
associated with tumor progression in breast cancer
Cristina Cadenas1*, Leonie van de Sandt2, Karolina Edlund1, Miriam Lohr2,
Birte Hellwig2, Rosemarie Marchan1, Marcus Schmidt3, Jörg
Rahnenführer2, Henrik Oster4 and Jan G. Hengstler1
Figure legends
Supplemental Figure 1: Association of circadian genes with metastasis free survival time. High expression of CLOCK
(217563_at), PER1 (202861_at), NPAS2 (205459_s_at) and RORC (206419_at) is significantly associated with longer
metastasis free survival. The analysis included 766 patients with node-negative breast cancer who have not been treated by
chemotherapy. All genes were dichotomized at the median. The log-rank test was used to assess statistical significance of the
Kaplan-Meier plots.
Supplemental Figure 2: Kaplan Meier plots showing metastasis survival rate of the different breast cancer subtypes,
classified according to (Desmedt et al., 2008). The most aggressive subtype is the ER-/HER2- whereas the least aggressive
subtype is the ER+/HER2-.
Supplemental Figure 3: Association of circadian genes with metastasis free survival time in the different breast
cancer molecular subtypes. A) In the ER+/HER2- breast cancer subtype (N=522) high expression of CRY2 (212696_at) is
significantly associated with longer metastasis free survival time whereas high expression of TIMELESS (203046_s_at) is
associated with shorter metastasis free survival. B) In the ER-/HER2- breast cancer subtype (N=140) both CLOCK
(217563_at) and NPAS2 (205459_s_at) are significantly associated with better outcome. C) ARNTL2 (220658_s_at) is
significantly associated with longer metastasis free survival in the HER2-overexpressing subgroup (N=104). All genes were
dichotomized at the median. The log-rank test was used to assess statistical significance of the Kaplan-Meier plots.
Supplemental Figure 4: Association of RNA levels of circadian genes with clinical parameters. A) For CLOCK
(217563_at) only significant association with lack of metastastatic occurrence and higher age was found. B) PER1 (36829_at)
associates with positive ER-status, negative HER2-status, lower grade, lack of metastasis, lower stage and higher age. C)
NPAS2 (205459_s_at) was found to associate with negative ER-status and lack of metastasis and D) RORC (206419_at)
behaved similar than PER1 except for lack of association with HER2-status. Differences for ER-status, HER2-status, pTstage,
metastasis and age were tested by the Mann-Whitney test; differences between grading by the Kruscal-Wallis test. The
analysis included 766 patients with node-negative breast cancer who have not been treated by chemotherapy.
Figure legends (continued)
Supplemental Figure 5: Rational for studying correlations among clock genes in breast cancer. In all panels, the
graphs at the left side represent the hypothetical expression levels of two circadian genes plotted against time. Day time is
shown as a white bar (circadian/Zeitgeber times 0h to 12h), night time as black bar (circadian/Zeitgeber times 12h to 24h).
The arrows indicate assumed time points of surgery for tumor tissue resection (usually at day time). These data points
were taken to plot expression of gene 1 against expression of gene 2 (graphs at the right side) and correlation coefficients
were calculated. Different scenarios are simulated. A: Two clock genes cycling in phase should result in a strong
correlation between their RNA levels. B: A different phase-relationship between the two genes is expected to result in a
lower correlation coefficient. C: Expression of two clock genes in anti-phase should yield a strong inverse correlation. D-E:
Reduced levels of expression of either both (D), or one of the genes (E) do not necessarily result in loss of correlation as
long as they are synchronized. F: Correlations may be lost due to loss of circadian rhythmicity.
Supplemental Figure 6: Heatmap of correlations of core clock components. A correlation matrix of Spearman´s rank
correlation coefficients among circadian genes is displayed as a heatmap. With exception of the perfect correlations in the
diagonal (clock genes with themselves) and the two PER1 probe sets with each other, correlations between PER2
(205251_at) and PER3 (221045_s_at) and CRY2 (212695_at) and PER3 (221045_s_at) were found to be the highest (R=
> 0.5) in the combined cohort of 766 node-negative breast cancer patients.
Supplemental Figure 7: Correlations of circadian clock genes in tumors stratified according to ER-status, HER2status, histological grade and metastasis occurrence. The pairwise correlation analysis shown in Fig. 4 for the
combined cohort was performed as well in the Mainz (A and B), TRANSBIG (C and D) and Rotterdam (E and F) cohorts.
Correlation coefficients between PER2 (205251_at) and PER3 (221045_s_at) and between CRY2 (212695_at) and PER3
(221045_s_at) are higher in estrogen receptor (ER) positive and HER2 status negative tumors, and also in tumors with
low grade and with no metastatic occurrence (green color). Conversely, correlations are lower in ER negative, HER2
status positive, high grade and metastatic tumors (red color). Correlations were analyzed using the Spearman correlation
test.
Supplemental Figure 8: Evaluation of the prognostic value of lack of coordinated expression of clock genes. The
residual values in the regression plot of PER2-PER3 correlations were calculated and the association of absolute residual
with prognosis was evaluated in the univariate and multivariate Cox analysis. The absolute residual showed a trend to a
higher hazard ratio but did not amount to statistical significance.
CLOCK > median
CLOCK < median
P= 0.013
Metastasis free survival rate
Metastasis free survival rate
Supplemental Fig. 1
NPAS2 > median
NPAS2 < median
P= 0.004
Metastasis free survival (years)
PER1 < median
P= 0.008
Metastasis free survival (years)
Metastasis free survival rate
Metastasis free survival rate
Metastasis free survival (years)
PER1 > median
RORC > median
RORC < median
P< 0.001
Metastasis free survival (years)
Metastasis free survival rate
Supplemental Fig. 2
Metastasis free survival (years)
Supplemental Fig. 3
A
C
E
N=522
P<0.001
Metastasis free survival (years)
B
CLOCK > median
CLOCK < median
N= 140
P = 0.003
Metastasis free survival (years)
D
TIMELESS > median
N=522
P<0.001
Metastasis free survival (years)
Metastasis free survival rate
Metastasis free survival rate
TIMELESS < median
NPAS2 > median
NPAS2 < median
N=140
P=0.013
Metastasis free survival (years)
Metastasis free survival rate
CRY2 < median
Metastasis free survival rate
Metastasis free survival rate
CRY2 > median
ARNTL2 > median
ARNTL2 < median
N= 104
P = 0.01
Metastasis free survival (years)
Supplemental Fig. 4
A
Supplemental Fig. 4 (continued)
B
Supplemental Fig. 4 (continued)
C
Supplemental Fig. 4 (continued)
D
Supplemental Fig. 5
A
D
B
E
C
F
Supplemental Fig. 6
Supplemental Fig. 7
A
ER+ vs. ERR=0.592
R=-0.024
B
ER+ vs. ERR=0.527
R=0.324
HER2- vs. HER2+
R=0.612
R=0.228
HER2- vs. HER2+
R=0.563
R=0.314
Grade 1+2 vs. Grade 3
R=0.557
R=0.216
Grade 1+2 vs. Grade 3
R=0.554
R=0.278
No metastasis vs.
early metastasis
R=0.618
R=0.286
No metastasis vs.
early metastasis
R=0.605
R=0.304
Supplemental Fig. 7 (continued)
C
ER+ vs. ERR=0.544
R=0.042
D
ER+ vs. ERR=0.488
R=0.198
HER2- vs. HER2+
R=0.554
R=-0.213
HER2- vs. HER2+
R=0.534
R=0.110
Grade 1+2 vs. Grade 3
R=0.535
R=0.143
Grade 1+2 vs. Grade 3
R=0.489
R=0.283
No metastasis vs.
early metastasis
R=0.487
R=0.261
No metastasis vs.
early metastasis
R=0.530
R=0.283
Supplemental Fig. 7 (continued)
E
ER+ vs. ERR=0.499
R=0.115
F
ER+ vs. ERR=0.349
R=0.086
HER2- vs. HER2+
R=0.533
R=0.641
HER2- vs. HER2+
R=0.430
R=0.315
No metastasis vs.
early metastasis
R=0.571
R=0.354
No metastasis vs.
early metastasis
R=0.461
R=0.372
Supplemental Fig. 8
Univariate Cox model
PER3 (221045_at) expression
Grade 1 vs. Grade 3 (Mainz)
R=0.658
R=0.216
variable
HR
P value
PER2
0.457
0.001
PER3
0.415
0.001
residual
0.785
0.395
abs. residual
1.724
0.155
Multivariate Cox model with PER2 and PER3
variable
HR
P value
PER2
0.633
0.133
PER3
0.517
0.030
Multivariate Cox model with PER2, PER3
and absolute residual
variable
HR
P value
PER2
0.624
0.097
PER3
0.539
0.032
abs. residual
1.340
0.441
PER2 (205251_at) expression