Upload
posy-sullivan
View
215
Download
0
Tags:
Embed Size (px)
Citation preview
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, HER2-status, 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.
Met
asta
sis
fre
e su
rviv
al r
ate
Metastasis free survival (years)
Met
asta
sis
fre
e su
rviv
al r
ate
Met
asta
sis
fre
e su
rviv
al r
ate
Metastasis free survival (years)
Met
asta
sis
fre
e su
rviv
al r
ate
Metastasis free survival (years)
CLOCK > median
CLOCK < median
PER1 > median
PER1 < median
NPAS2 > median
NPAS2 < median
RORC > median
RORC < median
Metastasis free survival (years)
P= 0.013 P= 0.008
P= 0.004 P< 0.001
Supplemental Fig. 1
Met
asta
sis
fre
e su
rviv
al r
ate
Metastasis free survival (years)
Supplemental Fig. 2
Met
asta
sis
fre
e s
urvi
val r
ate
Metastasis free survival (years)
Met
asta
sis
fre
e s
urvi
val r
ate
Metastasis free survival (years)
Met
asta
sis
fre
e s
urvi
val r
ate
Metastasis free survival (years)M
etas
tasi
s fr
ee s
urvi
val r
ate
Metastasis free survival (years)M
etas
tasi
s fr
ee s
urvi
val r
ate
Metastasis free survival (years)
Supplemental Fig. 3
A
B
C
CRY2 > median
CRY2 < median
N=522 P<0.001
CLOCK > median
CLOCK < median
N= 140 P = 0.003
TIMELESS > median
TIMELESS < median
N=522 P<0.001
NPAS2 > median
NPAS2 < median
N=140 P=0.013
ARNTL2 > median
ARNTL2 < median
N= 104 P = 0.01
D
E
Supplemental Fig. 4
A
Supplemental Fig. 4 (continued)
B
Supplemental Fig. 4 (continued)
C
Supplemental Fig. 4 (continued)
D
A
B
C
D
E
F
Supplemental Fig. 5
Supplemental Fig. 6
ER+ vs. ER- HER2- vs. HER2+
HER2- vs. HER2+ER+ vs. ER- Grade 1+2 vs. Grade 3
No metastasis vs. early metastasis
No metastasis vs. early metastasis
Grade 1+2 vs. Grade 3
R=0.592R=-0.024
R=0.612R=0.228
R=0.557R=0.216
R=0.618R=0.286
R=0.527R=0.324
R=0.563R=0.314
R=0.554R=0.278
R=0.605R=0.304
Supplemental Fig. 7
A
B
ER+ vs. ER- HER2- vs. HER2+
HER2- vs. HER2+
Grade 1+2 vs. Grade 3
ER+ vs. ER- Grade 1+2 vs. Grade 3
No metastasis vs. early metastasis
No metastasis vs. early metastasis
R=0.544R=0.042
R=0.554R=-0.213
R=0.535R=0.143
R=0.487R=0.261
R=0.488R=0.198
R=0.534R=0.110
R=0.489R=0.283
R=0.530R=0.283
Supplemental Fig. 7 (continued)
C
D
ER+ vs. ER- HER2- vs. HER2+
HER2- vs. HER2+ER+ vs. ER-
No metastasis vs. early metastasis
No metastasis vs. early metastasis
R=0.499R=0.115
R=0.533R=0.641
R=0.571R=0.354
R=0.349R=0.086
R=0.430R=0.315
R=0.461R=0.372
E
F
Supplemental Fig. 7 (continued)
R=0.658 R=0.216
Grade 1 vs. Grade 3 (Mainz)
PER2 (205251_at) expression
PE
R3
(221
045
_at)
ex
pre
ssio
nvariable HR P value
PER2 0.457 0.001
PER3 0.415 0.001
residual 0.785 0.395
abs. residual 1.724 0.155
variable HR P value
PER2 0.633 0.133
PER3 0.517 0.030
variable HR P value
PER2 0.624 0.097
PER3 0.539 0.032
abs. residual 1.340 0.441
Univariate Cox model
Multivariate Cox model with PER2 and PER3
Multivariate Cox model with PER2, PER3 and absolute residual
Supplemental Fig. 8