Relationships between CIN and prognostic factors of histopathology in EC
To investigate the CIN reflected by CIN signatures in EC, we first confirmed the difference in CIN signatures between benign and malignant endometria. In the TCGA Uterine Corpus Endometrial Carcinoma (UCEC) cohort, 23 cancer samples had notably increased CIN25 and CIN70 expression levels compared to matched adjacent normal tissues (CIN25: p < 0.001; CIN70: p < 0.001; Figure S1A). Analysis in the GSE63678 dataset, which contained endometrioid EC (EEC) and four rare pathological types (mixed carcinoma with villoglandular, squamous differentiation, clear cell or papillary serous) gave similar results (CIN25: p = 0.003; CIN70: p = 0.003; Figure S1B). Additionally, in the GSE17025 dataset, ECs had significantly increased CIN25 and CIN70 compared with benign lesions of the endometrium, including polyps and atrophic, inactive or cystic endometria (CIN25: p < 0.001; CIN70: p < 0.001; Figure S1C).
The nearly identical outcomes of these detections indicated that abnormal chromosomal stability represented by elevated CIN signatures was a dominant feature of EC. For further exploration of CIN in EC, we then compared CIN signatures among prognostic factors of histopathology.
First, in the TCGA UCEC cohort, the highest, intermediate and lowest CIN25 and CIN70 values were found in Grade 3, Grade 2 and Grade 1 patients, respectively (CIN25: p < 0.001; CIN70: p < 0.001; Figure 1A). Meta-analysis including 483 Grade 1 & 2 and 478 Grade 3 patients from 9 EC datasets confirmed the aggravated CIN in Grade 3 (CIN25 SMD: 0.985, 95% confidence interval (CI): 0.85 to 1.13, p = 0.000; CIN70 SMD: 1.009, 95% CI: 0.87 to 1.15, p = 0.000; Figure 1B and Figure S1D). This finding suggested that the more serious the CIN, the poorer the tumor differentiation. Second, we observed low-level expression of CIN signatures in EECs and high-level expression in non-EECs from TCGA (CIN25: p < 0.001; CIN70: p < 0.001; Figure 1C). That unfavorable histopathological type of EC tended to have severe CIN was further demonstrated by meta-analysis comprising 710 EEC and 253 non-EEC patients from 9 datasets (CIN25 SMD: 0.69, 95% CI: 0.54 to 0.84, p = 0.000; CIN70 SMD: 0.63, 95% CI: 0.48 to 0.78, p = 0.000; Figure 1D and Figure S1E). Third, a meta-analysis of 447 Stage Ⅰ & Ⅱ patients versus 184 Stage Ⅲ & Ⅳ patients from 4 EC datasets showed that Stage Ⅲ & Ⅳ patients had obviously increased CIN signatures (CIN25 SMD: 0.602, 95% CI: 0.43 to 0.78, p = 0.000; CIN70 SMD: 0.592, 95% CI: 0.42 to 0.77, p = 0.000; Figure 1E and Figure S1F). Furthermore, patients with longer distances of lymph node metastasis (aortic) or deeper MI (MI > 50%) in TCGA had much higher CIN25 and CIN70 (CIN25: p < 0.05; CIN70: p < 0.05; Figure 1F and 1G). Thus, the variation in CIN was also an important characteristic of EC progression. Finally, we detected significantly positive correlations between diagnosis age and CIN signatures in EEC patients with Stage Ⅰ, Grades 1 & 2 and MI < 50% from TCGA (CIN25: R2 = 0.05, p = 0.010; CIN70: R2 = 0.05, p = 0.011; Figure 1H). Analysis of the GSE17025 dataset yielded similar results (CIN25: R2 = 0.20, p = 0.050; CIN70: R2 = 0.25, p = 0.025; Figure 1I). Patients older than 60 tended to have elevated CIN25 and CIN70 compared with younger patients in TCGA (CIN25: p = 0.0050; CIN70: p = 0.0054; Figure 2J left). This trend between the two age subgroups did not reach a level of statistical significance in GSE17025, possibly due to insufficient samples (Figure 2J left).
Relationships between CIN and prognostic factors of molecular pathology in EC
As all unfavorable prognostic factors of histopathology are tightly associated with aggravated CIN, we speculated whether CIN signatures could be used to conduct risk assessments for different patients in the same adjuvant radiotherapy subgroup classified by the guidelines (observation (OB) subgroup, vaginal brachytherapy (VBT) subgroup and external beam radiation therapy (EBRT) subgroup; Materials and Methods and Table 1), thus providing some opportunities to further optimize indications for postoperative adjuvant therapy. Although patients with a high risk of recurrence or progression tended to have high CIN signatures, the areas under the curve (AUCs) for 5-year disease-free survival (DFS) of the OB, VBT, EBRT and EBRT EEC subgroups were not more than 0.67 (Figure 2A), which was unsatisfactory and prompted us to investigate possible factors for weakening the predictive power of CIN signatures.
Table 1. Clinicopathologic parameters according to adjuvant radiotherapy classification in Stage Ⅰ patients of TCGA UCEC cohort.
|
Total
|
OB
|
VBT
|
EBRT
|
|
|
Prognostic factors
|
n = 294 (100%)
|
n = 123 (42%)
|
n = 92 (31%)
|
n = 79 (27%)
|
P
|
|
|
Age a
|
|
|
|
|
|
|
Mean (range)
|
64 (31~90)
|
60 (31~89)
|
66 (35~90)
|
68 (35~87)
|
0.000
|
ANOVA b
|
< 60
|
97 (33%)
|
65 (53%)
|
20 (22%)
|
12 (15%)
|
0.000
|
Pearson Chi2
|
> 60
|
195 (67%)
|
58 (47%)
|
71 (78%)
|
66 (85%)
|
|
|
|
Histologic Type
|
|
|
|
|
0.000
|
Pearson Chi2
|
Type I, EEC
|
250 (85%)
|
123 (100%)
|
92 (100%)
|
35 (44%)
|
|
|
Type II, non-EEC
|
44 (15%)
|
0
|
0
|
44 (56%)
|
|
|
Grade
|
|
|
|
|
0.000
|
Pearson Chi2
|
1
|
76 (26%)
|
66 (54%)
|
10 (11%)
|
0
|
|
|
2
|
76 (26%)
|
57 (46%)
|
20 (22%)
|
1(1%)
|
|
|
3
|
140 (48%)
|
0
|
62 (67%)
|
78 (99%)
|
|
|
Stage c
|
|
|
|
|
0.000
|
Pearson Chi2
|
IA, MI < 50%
|
199 (68%)
|
105 (87%)
|
62 (67%)
|
32 (41%)
|
|
|
IB, MI > 50%
|
92 (32%)
|
16 (13%)
|
30 (33%)
|
46 (59 %)
|
|
|
CIN expression
|
|
|
|
|
|
|
CIN25 Mean (range)
|
-0.03 (-1.45~3.24)
|
-0.41 (-1.45~2.38)
|
0.18 (-1.20~3.24)
|
0.54 (-1.29~2.23)
|
0.000
|
ANOVA
|
CIN70 Mean (range)
|
-0.03 (-1.53~3.07)
|
-0.38 (-1.53~1.95)
|
0.17 (-1.09~3.07)
|
0.50 (-1.14~1.86)
|
0.000
|
ANOVA
|
Aneuploidy Score
|
4.82 (0~31)
|
2.35 (0~20)
|
4.38 (0~27)
|
9.18 (0~31)
|
0.000
|
ANOVA
|
FGA
|
0.15 (0~0.95)
|
0.08 (0~0.95)
|
0.15 (0~0.81)
|
0.25 (0~0.69)
|
0.000
|
ANOVA
|
|
|
|
|
|
|
|
|
|
|
Guidelines risk group d
|
|
|
|
|
0.000
|
Pearson Chi2
|
Low
|
105 (36%)
|
105 (87%)
|
0
|
0
|
|
|
Intermediate
|
46 (16%)
|
16 (13%)
|
30 (33%)
|
0
|
|
|
High-intermediate
|
62 (21%)
|
0
|
62 (67%)
|
0
|
|
|
High
|
79 (27%)
|
0
|
0
|
79 (100%)
|
|
|
TCGA Subtype
|
|
|
|
|
0.000
|
Pearson Chi2
|
POLE-mutant
|
29 (10%)
|
12 (10%)
|
10 (11%)
|
7 (9%)
|
|
|
MSI
|
99 (34%)
|
39 (32%)
|
37 (40%)
|
23 (29%)
|
|
|
CNV Low
|
101 (34%)
|
67 (54%)
|
25 (27%)
|
9 (11%)
|
|
|
CNV High
|
65 (22%)
|
5 (4%)
|
20 (22%)
|
40 (51%)
|
|
|
Mutation
|
|
|
|
|
|
|
PTEN
|
223 (76%)
|
111 (90%)
|
73 (79%)
|
39 (49%)
|
0.000
|
Pearson Chi2
|
FGFR2
|
59 (20%)
|
23 (19%)
|
22 (24%)
|
14 (18%)
|
n.s.
|
Pearson Chi2
|
CTNNB1
|
72 (24%)
|
44 (36%)
|
22 (24%)
|
6 (8%)
|
0.000
|
Pearson Chi2
|
PIK3CA
|
147 (50%)
|
67 (54%)
|
47 (51%)
|
33 (42%)
|
n.s.
|
Pearson Chi2
|
PPP2R1A
|
40 (14%)
|
5 (4%)
|
12 (13%)
|
23 (29%)
|
0.000
|
Pearson Chi2
|
a For the two cases without age, one was in VBT group, another was in EBRT group; b One-way analysis of variance; c For the three cases without accurate MI, two were in OB group, one was in EBRT group; d There were two cases in OB group without complete clinicopathological information for guidelines risk assessment
Prognostic factors of molecular pathology became the focus of our investigation. Among the TCGA molecular subtypes of EC except POLE-mutant, CNV-L, MSI and CNV-H had the lowest, intermediate and highest risks of recurrence, respectively, and correspondingly had the lowest, intermediate and highest CIN25 and CIN70 (CIN25: p < 0.001; CIN70: p < 0.001; Figure 2B and 2C) (5, 29, 30), which once again implied that CIN might positively correlate with the risk of recurrence in EC. The only exceptional subtype was POLE-mutant, whose prognosis was the best among the four TCGA molecular subtypes, but its CIN signature expression was comparable to that of CNV-H, which had the worst outcome (CIN25: p > 0.05; CIN70: p > 0.05; Figure 2B and 2C) (5, 29, 30). This phenomenon inspired us to explore whether other mutations with prognostic value also had special CIN signatures and in which adjuvant radiotherapy subgroup these special CIN signatures existed. To this end, we compared CIN signatures in wild-type patients with those in POLE, CTNNB1, PTEN, PIK3CA, FGFR2 and PPP2R1A mutant patients from subgroups of OB, VBT, EBRT and ICGC PanCancer Analysis of Whole Genomes (PCAWG) (Figure 2D and Figure S2). POLE mutant patients in the OB and VBT subgroups did not relapse or die (Figure 2E) but had higher expression of CIN25 and CIN70 compared with wild-type patients (CIN25: p < 0.05; CIN70: p < 0.05; Figure 2D), which might interfere with the risk assessment of CIN signatures. In the OB and EBRT subgroups, the CTNNB1 mutation was another special mutation that had much lower CIN signatures (CIN25: p < 0.05; CIN70: p < 0.05; Figure 2D and Figure S2E) but had a much worse prognosis than the wide type (Figure 2F) (5, 31). Multivariable Cox models further demonstrated that CTNNB1 mutation was an unfavorable prognostic factor independent of CIN signatures in the OB and EBRT subgroups (Tables 2 and 3). However, this conclusion did not hold in the VBT subgroup, whose CIN signature expression was exactly similar between the CTNNB1 mutant and the wild type (Figure 2D and Table 2).
Table 2. Multivariable analysis on the prognosis role of CIN signatures and CTNNB1 mutation in OB and VBT subgroups without POLE mutation.
Disease-free survival
|
Overall survival
|
OB
|
n
|
HR (95% CI)
|
P
|
n
|
HR (95% CI)
|
P
|
CIN25
|
Low
|
61
|
1
|
33
|
1
|
High
|
50
|
2.295 (0.749-7.029)
|
0.146
|
78
|
-
|
0.97
|
CTNNB1
|
Wild type
|
67
|
1
|
67
|
1
|
Mutation
|
44
|
1.400 (0.473-4.138)
|
0.543
|
44
|
12.393 (1.325-99.433)
|
0.022
|
CIN70
|
Low
|
24
|
1
|
44
|
1
|
High
|
87
|
-
|
0.958
|
67
|
-
|
0.966
|
CTNNB1
|
Wild type
|
67
|
1
|
67
|
1
|
Mutation
|
44
|
1.576 (0.545-4.554)
|
0.401
|
44
|
12.289 (1.431-105.564)
|
0.022
|
VBT
|
n
|
HR (95% CI)
|
P
|
|
n
|
HR (95% CI)
|
P
|
CIN25
|
Low
|
62
|
1
|
63
|
1
|
High
|
20
|
6.183 (1.416-26.991)
|
0.015
|
19
|
2.644 (0.372-18.807)
|
0.331
|
CTNNB1
|
Wild type
|
60
|
1
|
60
|
1
|
Mutation
|
22
|
0.562 (0.065-4.838)
|
0.6
|
22
|
-
|
0.978
|
CIN70
|
Low
|
50
|
1
|
55
|
1
|
High
|
32
|
6.032 (1.215-29.949)
|
0.028
|
27
|
2.311 (0.325-16.422)
|
0.403
|
CTNNB1
|
Wild type
|
60
|
1
|
60
|
1
|
Mutation
|
22
|
0.307 (0.038-2.500)
|
0.27
|
22
|
-
|
0.976
|
Table 3. Multivariable analysis on the prognosis role of CTNNB1 and POLE mutations and CIN signatures in EBRT subgroup.
|
|
Disease-free survival
|
|
|
|
Overall survival
|
|
EBRT
|
n
|
HR (95% CI)
|
P
|
|
n
|
HR (95% CI)
|
P
|
CIN25
|
|
|
|
|
|
|
|
Low
|
30
|
1
|
|
|
11
|
1
|
|
High
|
49
|
2.772 (0.735-10.459)
|
0.132
|
|
68
|
-
|
0.977
|
CTNNB1
|
|
|
|
|
|
|
|
Wild type
|
73
|
1
|
|
|
73
|
1
|
|
Mutation
|
6
|
4.907 (1.008-23.880)
|
0.049
|
|
6
|
6.654 (1.280-34.586)
|
0.024
|
POLE
|
|
|
|
|
|
|
|
Wild type
|
72
|
1
|
|
|
72
|
1
|
|
Mutation
|
7
|
0.740 (0.093-5.872)
|
0.776
|
|
7
|
-
|
0.983
|
CIN70
|
|
|
|
|
|
|
|
Low
|
33
|
1
|
|
|
12
|
1
|
|
High
|
46
|
3.039 (0.812-11.369)
|
0.092
|
|
67
|
-
|
0.976
|
CTNNB1
|
|
|
|
|
|
|
|
Wild type
|
73
|
1
|
|
|
73
|
1
|
|
Mutation
|
6
|
4.889 (1.013-23.602)
|
0.048
|
|
6
|
6.494 (1.249-33.759)
|
0.026
|
POLE
|
|
|
|
|
|
|
|
Wild type
|
72
|
1
|
|
|
72
|
1
|
|
Mutation
|
7
|
0.727 (0.092-5.755)
|
0.763
|
|
7
|
-
|
0.983
|
CIN signatures were prognostic in different adjuvant radiotherapy subgroups of EC
Consequently, we tested the prognostic value of CIN signatures in different adjuvant radiotherapy subgroups excluding different special mutations. For the OB subgroup without POLE and CTNNB1 mutations, the AUC based on CIN70 was 0.76 (Figure 3A), and the CIN70 High group predicted worse DFS than the CIN70 Low group (Figure 3B). For POLE wild types from the VBT subgroup, the AUC based on CIN25 was 0.71 (Figure 3A), and the CIN25 High group had a much lower 5-year DFS rate than the CIN25 Low group (Figure 3C). For CTNNB1 wild types from EBRT and EBRT EEC patients, the AUCs based on CIN25 were 0.62 and 0.72 (Figure 3A), and the outcomes of the CIN25 High group were much worse than those of the CIN25 Low group (Figure 3D and 3E). The predictive powers of the Fraction Genome Altered (FGA) and Aneuploidy Score, two signatures that only evaluate chromosomal content, were far inferior to that of CIN signatures (Figure 3A). Recurrent patients belonging to different histopathological types or TCGA molecular subtypes can be effectively evaluated by CIN signatures in different adjuvant radiotherapy subgroups (Figure 3F).
Since the AUCs based on CIN70 for DFS and OS of CTNNB1-mutant patients from the OB subgroup were 0.71 and 0.72 (Figure 3G), we were curious whether CIN could also play a role in the risk assessment of these patients. Although no statistically significant association between the CIN70 Low group and the CIN70 High group was observed, patients with sufficiently long follow-up in the CIN70 High group exhibited a trend toward worse 5-year DFS (Figure 3H left). We extended our analysis to 10-year OS and found that the outcome of the CIN70 High group was much worse than that of the CIN70 Low group (Figure 3H right). We therefore reasoned that the CIN signature could and should be used to stratify the CTNNB1-mutant patients from the OB subgroup.
Integrated risk assessment for Stage Ⅰ EEC from TCGA
According to the different effects of CIN signatures, mutations and pathology, a risk assessment model integrating all these factors is proposed in Figure 4A for Stage Ⅰ EEC. In this model, four risk profiles (low, intermediate, high and ultrahigh risk) with different prognoses were considered suitable to receive OB, VBT, EBRT and radiotherapy in combination with systemic therapy after surgery. Among the different existing risk stratification systems, our integrated risk model had the highest AUCs for both DFS and OS (AUC for DFS = 0.75, AUC for OS = 0.76; Figure 4B) and was the only system that had significant prognostic value for both DFS and OS (Figure 4C and 4D; Figure S3).