Characteristics and survival in patients enrolled
We enrolled 200 patients with ECA for analysis, comprising 185 with HPVA and 15 with NHPVA (Table 1). Across both groups, the median follow-up period was 68 months (range: 1.5–125.1 months). Moreover, the 1-, 3-, and 5-year OS rates were 99.5%, 90.5%, 85.0%, respectively.
Table 1. Patient demographics and clinical charateristics.
|
Characteristic
|
Total (n=200)
|
HPVA (n=186)
|
NHPVA (n=16)
|
|
|
No. (%)
|
No. (%)
|
No. (%)
|
Pa
|
Age
|
|
|
|
0.014
|
≤ 37
|
34 (17.0%)
|
28 (82.4%)
|
6 (17.6%)
|
|
> 37
|
166 (83.0%)
|
157 (94.6%)
|
9 (5.4%)
|
|
Serum HPV DNA
|
|
|
|
0.000
|
Negative (0-1pg/ml)
|
34 (17.0%)
|
26 (76.5%)
|
8 (23.5%)
|
|
Positive (≥ 1pg/ml)
|
110 (55.0%)
|
108 (98.2%)
|
2 (1.8%)
|
|
Not avaiable
|
56 (28.0%)
|
51 (51.8%)
|
5 (4.2%)
|
|
HPV genotype
|
|
|
|
0.273
|
HPV 16
|
49 (24.5%)
|
47 (95.9%)
|
2 (4.1%)
|
|
HPV 18
|
60 (30.0%)
|
57 (95.5%)
|
3 (5.0%)
|
|
Other
|
11 (5.5%)
|
11 (100.0%)
|
0 (0.0%)
|
|
Negative
|
72 (36.0%)
|
63 (87.5%)
|
9 (12.5%)
|
|
Not avaiable
|
8 (4%)
|
7 (87.5%)
|
1 (12.5%)
|
|
HPV RNAscope
|
|
|
|
0.003
|
≤ 3.3
|
99 (49.5%)
|
86 (46.5%)
|
13 (13.1%)
|
|
> 3.3
|
101 (50.5%)
|
99 (98.0%)
|
2 (2.0%)
|
|
Tumor size (cm)
|
|
|
|
0.004
|
≤ 4.5
|
178 (89.0%)
|
168 (94.4%)
|
10 (5.6%)
|
|
> 4.5
|
22 (11.0%)
|
17 (77.3%)
|
5 (22.7%)
|
|
FIGO stage
|
|
|
|
0.000
|
I
|
141 (70.5%)
|
137 (97.2%)
|
4 (2.8%)
|
|
II
|
51 (25.5%)
|
43 (84.3%)
|
8 (15.7%)
|
|
III
|
5 (2.5%)
|
4 (80.0%)
|
1 (20.0%)
|
|
IV
|
3 (1.5%)
|
1 (33.3%)
|
2 (66.7%)
|
|
Differentiation
|
|
|
|
0.537
|
Good
|
10 (5.0%)
|
7 (70.0%)
|
3 (30.0%)
|
|
Moderate
|
106 (53.0%)
|
96 (90.6%)
|
10 (9.4%)
|
|
Poor
|
84 (42.0%)
|
82 (97.6%)
|
2 (2.4%)
|
|
LVI
|
|
|
|
0.141
|
None (0)
|
138 (69.0%)
|
130 (94.2%)
|
8 (5.8%)
|
|
Focal (1-4)
|
37 (18.5%)
|
31 (83.8%)
|
6 (16.2%)
|
|
Moderate (5-9)
|
15 (7.5%)
|
14 (93.3%)
|
1 (6.7%)
|
|
Extensive (≥10)
|
10 (66.7%)
|
10 (100.0%)
|
0 (0.0%)
|
|
Invasion level of uterine cervix
|
|
|
|
0.008
|
< 1/3
|
54 (27.0%)
|
53 (98.1%)
|
1 (1.9%)
|
|
1/3-2/3
|
61 (31.0%)
|
60 (96.8%)
|
2 (3.2%)
|
|
≥ 2/3
|
84 (42.0%)
|
72 (85.7%)
|
12 (14.3%)
|
|
Lymph nodes invasion
|
|
|
|
0.024
|
No
|
154 (77.0%)
|
146 (94.8%)
|
8 (5.2%)
|
|
Yes
|
46 (23.0%)
|
39 (84.8%)
|
7 (46.7%)
|
|
Parametrium invasion
|
|
|
|
0.599
|
No
|
181 (90.5%)
|
168 (92.8%)
|
13 (7.2%)
|
|
Yes
|
19 (9.5%)
|
17 (89.5%)
|
2 (10.5%)
|
|
Surgical margin
|
|
|
|
0.000
|
No
|
187 (93.5%)
|
177 (94.7%)
|
10 (5.3%)
|
|
Yes
|
13 (6.5%)
|
8 (61.5%)
|
5 (38.5%)
|
|
Treatment
|
|
|
|
0.235
|
Surgery
|
83 (41.5%)
|
80 (96.4%)
|
3 (3.6%)
|
|
Surgery+chemotherapy
|
21 (10.5%)
|
20 (95.2%)
|
1 (4.8%)
|
|
Surgery+radiotherapy
|
41 (20.5%)
|
36 (87.8%)
|
5 (12.2%)
|
|
Surgery+chemoradiation
|
55 (27.5%)
|
49 (89.1%)
|
6 (10.9%)
|
|
MMR status
|
|
|
|
0.978
|
dMMR
|
13 (6.5%)
|
12 (92.3%)
|
1 (7.7%)
|
|
pMMR
|
187 (93.5%)
|
173 (92.5%)
|
14 (7.5%)
|
|
P16 IHC
|
|
|
|
0.001
|
Negative
|
82 (41.0%)
|
70 (85.4%)
|
12 (14.6%)
|
|
Positive
|
118 (59.0%)
|
115 (97.5%)
|
3 (2.5%)
|
|
Ki-67 IHC
|
|
|
|
0.131
|
≤12.5%
|
48 (24%)
|
42 (87.5%)
|
6 (12.5%)
|
|
>12.5%
|
152 (76%)
|
143 (94.1%)
|
9 (5.9%)
|
|
aChi-square test. HPVA, HPV-associated adenocarcinoma; NHPVA, nonHPV-associated adenocarcinoma; IHC, immunohistochemistry; LVI, lymph vascular invasion; dMMR, delete mismatch repair; pMMR, proficient mismatch repair.
Diagnostic performance of HPV E6/E7 mRNA in situ hybridization in ECA compared to other assays.
In situ expression of HPV mRNA was detected using RNAscope and scored using the RNAscope scoring system. HPV mRNA was mainly detected in the cytoplasm of cancer cells, with variable staining intensity. The RNAscope scores of 0 to 4 are shown in Fig. 1A and 1B. The diagnostic implications of the HPV E6/E7 RNAscope scores were evaluated in 200 ECA samples that included cases of HPVA and NHPVA (Table S1). The positive rates of HPV PCR, p16 immunohistochemistry (IHC), HPV genotype, and HPV E6/E7 RNAscope across all cases of ECA were 76.4%, 59.0%, 62.5%, and 72.0%, respectively (Fig. 1C and Fig. S3). HPV RNAscope and other assays are closely related (Table S2). Receiver operating characteristics (ROC) curve analyses suggested that HPV DNA and HPV E6/E7 RNAscope showed similar results in terms of distinguishing HPVA from NHPVA (area under the curve [AUC] = 0.802, sensitivity = 80.5%, specificity = 80% vs. AUC = 0.799, sensitivity = 75.8%, specificity = 80%, respectively), outperforming both p16 IHC (AUC = 0.751, sensitivity = 60.2%, specificity = 90%) and HPV PCR (AUC = 0.566, sensitivity = 63.3%, specificity = 50%; Fig. 1D and Table 5).
The correlations among serum HPV DNA level, p16 IHC, HPV PCR, and HPV E6/E7 RNAscope was also studied (Table S2). A significantly higher proportion of ECA cases showed positivity for p16 IHC, HPV PCR, and HPV E6/E7 RNAscope in subgroups with HPV DNA levels that differed by 10-fold. In patients with serum HPV levels of 1–100 pg/mL, the positivity for p16 IHC was similar to that for HPV PCR (60%). In patients with serum HPV levels of 100–10,000 pg/mL, the positivity for HPV PCR was similar to that for HPV E6/E7 RNAscope (93.8%). The positivity for HPV E6/E7 RNAscope for the diagnosis of ECA was much higher than that of p16 IHC and HPV PCR (Fig. S4A). Our results also showed that HPV E6/E7 RNAscope was positive in almost all cases that showed p16 IHC positivity (88.5% of all cases, 89.5% of HPVA cases), but that more cases were negative by IHC and serum levels (Fig. S4B). In different HPV subtypes, the positivity of RNAscope for the diagnosis of ECA was much higher compared to that of p16 IHC, but lower compared to HPV DNA (Fig. S4C). The HPV subtypes in patients with HPVA and NHPVA are listed in Table S3. In cases with RNAscope scores of 3 and 4, the positivity for HPV DNA was higher than that for p16 IHC and HPV PCR (Fig. S4D).
Multivariate analysis of the OS
Univariate analysis indicated that the following variables were related to OS in patients with ECA: age (p =0.002), HPV E6/E7 RNAscope (p = 0.006), tumor size (p = 0.005), FIGO stage (p < 0.001), histological type (p = 0.004), LVI (p < 0.001), invasion level of uterine cervix (p < 0.001), LNI (p < 0.001), parametrium invasion (p < 0.001), surgical margin (p = 0.006), chemotherapy (p = 0.009), radiotherapy (p < 0.001), p16 (p = 0.039), and Ki-67 (p = 0.025). In multivariate analysis, the following items remained independently prognostic: age (p = 0.003, HR = 0.250, 95% confidence interval [CI]: 0.099–0.632), HPV E6/E7 RNAscope (p = 0.003, HR=0.240, 95% CI: 0.093–0.616), LVI (p = 0.001, HR = 1.924, 95% CI: 1.295–2.857), LNI (p = 0.021, HR = 3.047, 95% CI: 1.183–7.849). The results are shown in Table 2, with a forest plot in Fig. 2A. Kaplan–Meier analysis showed significant diversity (Fig. 2B–E). Furthermore, the ROC curve for OS revealed that our model was higher than that of the FIGO stage and treatment (Fig. 3A).
Table 2. Univariate and multivariate Cox proportional hazards regression analysis for OS.
Variables
|
Univariate analysis
|
|
Multivariate analysis
|
HR (95% CI)
|
P value
|
|
HR (95% CI)
|
P value
|
Age (≤37 vs. >37 years)
|
0.316 (0.154-0.649)
|
0.002
|
|
0.224 (0.095-0.530)
|
0.001
|
Serum HPV DNA (negative vs. positive vs. na)
|
0.868 (0.500-1.509)
|
0.616
|
|
|
|
HPV genotype (negative vs. positive vs. na)
|
1.004 (0.523-1.926)
|
0.992
|
|
|
|
HPV RNAscope (≤3.3 vs. >3.3)
|
0.337 (0.156-0.730)
|
0.006
|
|
0.288 (0.124-0.673)
|
0.004
|
Tumor size (≤4.5 vs. >4.5 cm)
|
3.165 (1.414-7.083)
|
0.005
|
|
0.773 (0.223-2.674)
|
0.684
|
FIGO stage (I vs. II vs. III vs. IV)
|
2.821 (1.703-4.672)
|
0.000
|
|
1.120 (0.613-2.013)
|
0.705
|
Histological type (HPVA vs. NHPVA)
|
0.266 (0.108-0.657)
|
0.004
|
|
1.365 (0.334-5.589)
|
0.665
|
Differentiation (good vs. moderate vs. poor)
|
1.435 (0.768-2.684)
|
0.258
|
|
|
|
LVI (none vs. focal vs. moderate vs. extensive)
|
2.056 (1.508-2.803)
|
0.000
|
|
1.770 (1.212-2.587)
|
0.003
|
Invasion level of uterine cervix (1/3 vs. 1/3-2/3 vs. 2/3)
|
3.147 (1.754-5.648)
|
0.000
|
|
1.532 (0.791-2.967)
|
0.206
|
Lymph nodes invasion (no vs. yes)
|
9.310(4.533-19.120)
|
0.000
|
|
2.838 (1.162-6.931)
|
0.022
|
Parametrium invasion (no vs. yes)
|
0.231 (0.094-0.568)
|
0.001
|
|
0.696 (0.217-2.234)
|
0.543
|
Surgical margin (no vs. yes)
|
0.263 (0.101-0.687)
|
0.006
|
|
1.621 (0.435-6.048)
|
0.472
|
Treatment (s vs. s+ct vs. s+rt vs. s+cr)
|
1.733 (1.298-2.313)
|
0.000
|
|
1.182 (0.851-1.641)
|
0.318
|
MMR status (dMMR vs. pMMR)
|
1.401 (0.426-4.610)
|
0.579
|
|
|
|
P16 IHC (negative vs. positive)
|
0.478 (0.237-0.963)
|
0.039
|
|
1.203 (0.507-2.854)
|
0.675
|
Ki-67 IHC (≤12.5% vs. >12.5%)
|
0.446 (0.220-0.906)
|
0.025
|
|
0.841 (0.348-2.035)
|
0.701
|
HR, hazard ratio; CI, confidence interval. HPVA, HPV-associated adenocarcinoma; NHPVA, non HPV-associated adenocarcinoma; IHC, immunohistochemistry; LVI, lymph vascular invasion; dMMR, delete mismatch repair; pMMR, proficient mismatch repair; HPV genotype positive, HPV16, HPV18 and other types; s, surgery; s+ct, surgery+chemotherapy; s+rt, surgery+radiotherapy; s+cr, surgery+chemoradiation; na, not avaiable.
Comparison of efficiency between our nomogram and conventional systems.
A nomogram was built to predict OS. Model A included age, HPV E6/E7 RNAscope, LVI, and LNI; model B included age, HPV E6/E7 RNAscope, LVI, LNI, FIGO stage, and treatment (Fig. 3B and 3C). The results of comparison of efficiency between our nomogram and conventional systems are shown in Table 3. In the training group, no significant difference was observed (C-index: 0.825, 95% CI = 0.754–0.896 vs. 0.836, 95% CI = 0.771–0.902), the C-index of nomogram A (0.825, 95% CI = 0.754–0.896) was better than those of the FIGO system (0.653, 95% CI = 0.567–0.740) and treatment (0.578, 95% CI = 0.506–0.651). In Fig. 2A and 2B, the calibration plot for the OS rates after 1, 3, and 5-years showed agreement between the nomogram and actual observation. Besides, our model seemed to have higher prediction accuracy (Fig. 4C).
Table 3. The C-index of our model, FIGO stage, Treatment for prediction of OS in the ECA.
|
Factors
|
C-index
|
95 CI%
|
P
|
Nomogram A
|
0.825
|
0.754-0.896
|
|
FIGO stage
|
0.653
|
0.567-0.740
|
|
Treatment
|
0.578
|
0.506-0.651
|
|
Nomogram B
|
0.836
|
0.771-0.902
|
|
Nomogram A vs FIGO stage
|
|
|
0.002
|
Nomogram A vs Treatment
|
|
|
<0.001
|
Nomogram A vs Nomogram B
|
|
|
0.139
|
Nomogram A: age + HPV RNA scope + LVI + Lymph nodes invasion
Nomogram B: age + HPV RNA scope + LVI + Lymph nodes invasion+ FIGO stage+ Treatment
C-index = concordance index; LVI, lymph vascular invasion; P values are calculated based on normal approximation using function rcorrp.cens in Hmisc package.
Risk stratification of prognosis
All patients were divided into a low-risk group (≤ 15.75) and a high-risk group (> 15.75) for OS (Fig. S2B). The 1-, 3-, and 5-year OS rates between the two risk groups were 94.2%, 84.3%, 70.3%, respectively, in the low-risk group and 71.4%, 46.4%, 10.7%, respectively, in the high-risk group (Table 4). Moreover, significant differences in OS were noted between patients with stage I/II ECA and those with III/IV ECA (Fig. 5). Each risk subgroup represented a distinct prognosis, and our system accurately separated OS in the two subgroups.
Table 4. Point Assignment and Prognostic Score of the nomogram model
|
Variables
|
Prognostic Score
|
1-Year OS (%)
|
3-Year OS (%)
|
5-Year OS (%)
|
Age (years)
|
|
|
|
|
≤ 37
|
7.5
|
11.8
|
26.5
|
35.3
|
> 37
|
0
|
1.8
|
6.0
|
10.8
|
HPV RNA scope
|
|
|
|
|
≤ 3.3
|
7
|
5.1
|
13.1
|
21.2
|
> 3.3
|
0
|
2.0
|
5.9
|
8.9
|
Lymph nodes invasion
|
|
|
|
|
No
|
0
|
1.9
|
4.5
|
7.1
|
Yes
|
8.25
|
8.7
|
26.1
|
41.3
|
Lymph vascular invasion
|
|
|
|
|
None
|
0
|
1.4
|
6.5
|
10.1
|
Focal
|
3.25
|
2.7
|
10.8
|
13.5
|
Moderate
|
6.5
|
13.3
|
20.0
|
26.7
|
Extensive
|
9.75
|
20.0
|
30.0
|
70.0
|
Total prognostic Score
|
|
|
|
|
≤ 15.75
|
|
94.2
|
84.3
|
70.3
|
> 15.75
|
|
71.4
|
46.4
|
10.7
|
Table 5. Diagnostic performances of studied testing for ECA patients.
|
HPVA vs. NHPVA
|
AUC
|
95%CI
|
Sensitivity
|
Specificity
|
|
P16 IHC
|
0.751
|
0.620-0.882
|
0.602
|
0.900
|
|
HPV DNA
|
0.802
|
0.653-0.951
|
0.805
|
0.800
|
|
HPV genotype
|
0.566
|
0.379-0.754
|
0.633
|
0.500
|
|
HPV RNAscope
|
0.799
|
0.629-0.929
|
0.758
|
0.800
|
|
AUC, area under curve; 95%CI, 95% confident interval; HPVA, HPV-associated adenocarcinoma; NHPVA, nonHPV-associated adenocarcinoma; IHC, immunohistochemistry; HPV RNAscope.