Patient characteristics
A flow diagram of the study is provided in Figure 1. From June 1, 2019, to September 1, 2019, 368 eligible patients were recruited, and 307 were included in the final analysis. The median age was 46 years (range 22 to 77). Regarding primary diagnoses, there were 41 cases of benign ovarian or uterine disease, 101 of CIN2/3, 66 of cervical cancer, and 99 of ovarian or uterine malignancies, precancerous lesions, or borderline tumors (Table 1 and Supplement 1). The surgeries comprised 84 cases of conization or LEEP and 223 hysterectomies. The final cervical pathology results are listed in Table 1 and Figure 1. The median interval from biopsy to conization/LEEP or from conization/LEEP to hysterectomy/radical hysterectomy was 40 days (range 10 to 51).
Table 1
Cervical procedures and pathology before and after sampling for DNA methylation. Three cases were excluded from the table, including one cervical mucinous adenocarcinoma, one uterine clear cell carcinoma, and one vaginal melanoma. ADC, adenocarcinoma. CIN, cervical intraepithelial lesion. LEEP, loop electrosurgical excision procedure. SCC, squamous cell carcinoma.
Primary diagnosis
|
Cervical procedures before DNA methylation (n)
|
Surgeries after DNA methylation (n)
|
Actual cervical pathology after DNA methylation
|
None
|
Biopsy
|
Conization or LEEP
|
Conization or LEEP
|
Hysterectomy
|
Benign (n=41)
|
41
|
0
|
0
|
0
|
41
|
Inflammation (n=40)
CIN1 (n=1)
|
CIN2/3 (n=101)
|
0
|
84
|
17
|
84
|
17
|
Inflammation (n=16)
CIN1 (n=13)
CIN2 (n=17)
CIN3 (n=55)
|
Cervical carcinomas (n=66)
|
0
|
37
|
29
|
0
|
65
|
Inflammation (n=21)
CIN1 (n=1)
SCC (n=25)
ADC, villoglandular type (n=3)
ADC, in situ (n=6)
ADC, endocervical type (n=9)
ADC, mucinous type (n=1)
|
Others* (n=99)
|
99
|
0
|
0
|
0
|
99
|
Inflammation (n=96)
CIN1 (n=1)
Endometrial clear cell carcinoma (n=1)
Vaginal melanoma (n=1)
|
* Others includes gynecological malignancies, precancerous lesions, or borderline tumors rather than cervical lesions.
DNA methylation and hrHPV assays were successfully performed for all 307 patients. There were 68 (22.1%), 103 (33.6%), 109 (35.5%), 131 (42.7%) and 72 (23.5%) cases with positive EPB41L3, JAM3, EPB41L3 or JAM3, hrHPV and methylation plus hrHPV, respectively. The details are listed in Supplementary Table 1. In the whole cohort, patients with positive and negative methylation had similar ages (45.9±10.0 versus 47.9±12.1 years, p=0.143); however, patients with positive hrHPV were significantly younger than patients with negative hrHPV (42.8±10.5 versus 50.4±11.1 years, p<0.001).
Diagnostic accuracies for cervical lesions
The diagnostic accuracies of the various screening methods are listed in Table 2. The positive methylation status rates for normal pathology in the whole cohort and in the cohort with exclusions for gynecological malignancies, precancerous lesions, and borderline tumors were 26.0% (25/96) and 3.9% (3/77), respectively (Table 2). Therefore, the normal pathology of benign diseases served as a reference. As shown in Table 2, methylation, hrHPV status and their combination were all significantly related to the severity of the cervical lesion. However, compared with the reference, DNA methylation and methylation plus hrHPV achieved only marginal differences in CIN2 (with p values of 0.055 and 0.065), probably due to the small sample size for this pathology.
Table 2
The diagnostic accuracies of DNA methylation, hrHPV and their combinations for the final cervical pathology. ADC, adenocarcinoma. CIN2+, cervical intraepithelial neoplasia (CIN) 2 or more severe lesion. hrHPV, high-risk human papillomavirus. N/A, not available. NPV, negative predictive value. OR, odds ratio. 95% CI, 95% confidence interval. PPV, positive predictive value. SCC, squamous cell carcinoma.
Cervical pathology
|
DNA methylation
|
hrHPV
|
HPV16 or HPV18
|
DNA methylation + hrHPV
|
|
n (%)
|
OR (95% CI)
|
p
|
n (%)
|
OR (95% CI)
|
p
|
n (%)
|
OR (95% CI)
|
p
|
n (%)
|
OR (95% CI)
|
p
|
Normal in benign diseases* (n=77)
|
3 (3.9)
|
Reference
|
-
|
18 (23.4)
|
Reference
|
-
|
11 (14.3)
|
Reference
|
-
|
1 (1.3)
|
Reference
|
-
|
Normal in others† (n=96)
|
25 (26.0)
|
8.685 (2.511-30.405)
|
0.001
|
3 (3.1)
|
0.106 (0.030-0.375)
|
<0.001
|
0 (0.0)
|
0.000 (0.000-N/A)
|
0.996
|
0 (0.0)
|
0.000 (0.000-N/A)
|
0.997
|
CIN1 (n=15)
|
0 (0.0)
|
0.000 (0.000-N/A)
|
0.999
|
10 (66.7)
|
6.556 (1.982-21.683)
|
0.002
|
5 (33.3)
|
3.000 (0.860-10.460)
|
0.085
|
0 (0.0)
|
0.000 (0.000-N/A)
|
0.999
|
CIN2 (n=17)
|
3 (17.6)
|
5.286 (0.966-28.911)
|
0.055
|
13 (76.5)
|
10.653 (3.087-36.764)
|
<0.001
|
6 (35.3)
|
3.273 (1.004-10.671)
|
0.049
|
2 (11.8)
|
10.133 (0.863-119.205)
|
0.065
|
CIN3 (n=55)
|
38 (69.1)
|
55.137 (15.205-199.941)
|
<0.001
|
50 (90.9)
|
32.778 (11.356-94.611)
|
<0.001
|
33 (60.0)
|
9.000 (3.902-20.759)
|
<0.001
|
35 (63.6)
|
133.000 (17.158-1030.948)
|
<0.001
|
SCC (n=25)
|
24 (96.0)
|
592.00 (58.790-5961.242)
|
<0.001
|
22 (88.0)
|
24.037 (6.443-89.682)
|
<0.001
|
21 (84.0)
|
31.500 (9.068-109.428)
|
<0.001
|
22 (88.0)
|
557.333 (55.190-5628.193)
|
<0.001
|
ADC (n=18)
|
14 (77.8)
|
86.333 (17.392-428.568)
|
<0.001
|
14 (77.8)
|
11.472 (3.353-39.255)
|
<0.001
|
12 (66.7)
|
12.000 (3.726-38.646)
|
<0.001
|
11 (61.1)
|
119.429 (13.385-1065.636)
|
<0.001
|
Normal* or CIN1 (n=92)
|
3 (3.3)
|
Reference
|
-
|
28 (30.4)
|
Reference
|
-
|
16 (17.4)
|
Reference
|
-
|
1 (1.1)
|
Reference
|
-
|
CIN2+ (n=115)
|
79 (68.7)
|
65.105 (19.294-219.611)
|
<0.001
|
99 (86.1)
|
14.143 (7.094-28.197)
|
<0.001
|
72 (62.6)
|
7.953 (4.118-15.362)
|
<0.001
|
70 (60.9)
|
141.556 (19.043-1052.225)
|
<0.001
|
Sensitivity (%)
|
68.7
|
86.1
|
62.6
|
60.9
|
Specificity (%)
|
96.7
|
69.6
|
82.6
|
98.9
|
PPV
|
0.963
|
0.780
|
0.818
|
0.986
|
NPV
|
0.712
|
0.800
|
0.639
|
0.669
|
* These cases excluded gynecological malignancies, precancerous lesions, or borderline tumors rather than cervical lesions.
† These cases only included gynecological malignancies, precancerous lesions, or borderline tumors rather than cervical lesions.
For the diagnosis of CIN2+, the sensitivities, specificities, PPVs and NPVs of the methylation assay in the previous training set [14] and in the current trial were all equal: 72.1% versus 68.7% (p=0.525), 91.5% versus 96.7% (p=0.120), 0.679 versus 0.712 (p=0.232), and 0.930 versus 0.963 (p=0.552), respectively.
For the diagnosis of CIN2+, assays for methylation assessment, hrHPV testing, HPV 16/18 testing and their combination all had favorable results, with ORs of 69.105 (95% CI 19.294-219.611), 14.143 (7.094-28.197), 7.953 (4.118-15.362) and 141.556 (19.043-1052.225), respectively (Table 2).
- The sensitivity values of methylation assessment and the combination were lower than that of hrHPV testing (68.7%, 60.9% versus 86.1, p = 0.002 and <0.001), but that of methylation assessment was similar to that of HPV 16/18 testing (68.7% versus 62.6, p=0.331).
- The specificity values of both methylation assessment and the combination were significantly higher than that of hrHPV testing (96.7% and 98.9% versus 0.696, p <0.001 and <0.001), and that of methylation assessment was also significantly higher than that of HPV 16/18 testing (96.7% versus 82.6%, p=0.002).
- The PPVs of both methylation assessment and the combination were significantly higher than that of hrHPV testing (0.963 and 0.986 versus 0.780, p <0.001 and <0.001), and that of methylation assessment was also significantly higher than that of HPV 16/18 (0.963 versus 0.818, p=0.003).
- The NPV of methylation assessment, but not the combination, was similar to that of hrHPV (0.712 and 0.669 versus 0.800, p = 0.158 and 0.039), and that of methylation assessment was also similar to that of HPV 16/18 (0.712 versus 0.639, p=0.221).
Even in patients with negative hrHPV results, DNA methylation assessment still had a significant discrepancy for CIN2+ (OR 39.857, 95% CI 7.137-222.577). In this population, the sensitivity, specificity, PPV and NPV were 56.3%, 96.9%, 0.818 and 0.899, respectively.
Further analysis of CIN2/3 and cervical squamous cell carcinoma (SCC) is presented in Table 3. Regarding SCC with or without residual lesions, significant differences in the methylation assay (p<0.001) and in hrHPV testing (p=0.004) were evident; regarding CIN2/3 with or without residual lesions, significant differences in the methylation assay (p<0.001) and in hrHPV testing (p=0.004) were evident. Regarding CIN2/3 and SCC without residual lesions after biopsy or excision, their methylation statuses were similar to those of benign uterine and ovarian lesions (p = 0.999 and 0.998). Regarding cervical diseases with or without residual lesions, the positive hrHPV ratios were all significantly higher than those of benign uterine and ovarian lesions (all p values less than 0.05, Table 3).
Table 3
The diagnostic accuracies of DNA methylation, hrHPV and their combination for cervical SCC and CIN2/3 with or without residual lesions. CIN, cervical intraepithelial neoplasia. hrHPV, high-risk human papillomavirus. N/A, not available. OR, odds ratio. 95% CI, 95% confidence interval. SCC, squamous cell carcinoma.
Cervical pathology
|
DNA methylation
|
hrHPV
|
DNA methylation + hrHPV
|
|
n (%)
|
OR (95% CI)
|
p
|
n (%)
|
OR (95% CI)
|
p
|
n (%)
|
OR (95% CI)
|
p
|
Normal* (n=41)
|
3 (7.3)
|
Reference
|
-
|
2 (4.9)
|
Reference
|
-
|
1 (2.4)
|
Reference
|
-
|
SCC without residual lesions (n=16)
|
0 (0.0)
|
0.000 (0.000-N/A)
|
0.999
|
7 (43.8)
|
15.167 (2.687-85.598)
|
0.002
|
0 (0.0)
|
0.000 (0.000-N/A)
|
0.999
|
SCC with residual lesions (n=25)
|
24 (96.1)
|
304.000 (29.871-3093.870)
|
<0.001
|
22 (88.0)
|
143.000(22.173-922.234)
|
<0.001
|
22 (88.0)
|
293.333 (28.764-2991.426)
|
<0.001
|
CIN2/CIN3 without residual lesions (n=29)
|
0 (0.0)
|
0.000 (0.000-N/A)
|
0.998
|
18 (62.1)
|
31.909 (6.399-159.128)
|
<0.001
|
0 (0.0)
|
0.000 (0.000-N/A)
|
0.998
|
CIN2/CIN3 with residual lesions (n=72)
|
41 (56.9)
|
16.753 (4.730-59.332)
|
<0.001
|
63 (87.5)
|
136.500 (28.021-664.933)
|
<0.001
|
37 (51.4)
|
42.286 (5.513-324.365)
|
<0.001
|
* These cases only included uterine or ovarian benign diseases
For the diagnosis of cervical ADC, methylation, hrHPV and their combination all had favorable results (Table 4). According to various subtypes of ADC, compared with uterine or ovarian benign diseases, methylation (OR 44.333, 95% CI 6.230-315.499), hrHPV (156.000, 12.575-1935.197) and their combination (80.000, 7.111-900.008) had the highest ORs in endocervical ADC, the most common pathology in this cohort. However, due to the limited sample size of ADC, the differences between the various subtypes of ADC were not significant.
Table 4
The diagnostic accuracies of DNA methylation, hrHPV and their combination for cervical adenocarcinoma. ADC, adenocarcinoma. hrHPV, high-risk human papillomavirus. N/A, not available. NPV, negative predictive value. OR, odds ratio. 95% CI, 95% confidence interval. PPV, positive predictive value. SCC, squamous cell carcinoma.
Cervical pathology
|
DNA methylation
|
hrHPV
|
DNA methylation + hrHPV
|
|
n (%)
|
OR (95% CI)
|
p
|
n (%)
|
OR (95% CI)
|
p
|
n (%)
|
OR (95% CI)
|
p
|
Normal* (n=41)
|
3 (7.3)
|
Reference
|
-
|
2 (4.9)
|
Reference
|
-
|
1 (2.4)
|
Reference
|
-
|
ADC, villoglandular type (n=3)
|
3 (100.0)
|
N/A (0.000-N/A)
|
0.999
|
2 (66.7)
|
39.000 (2.397-634.654)
|
0.010
|
2 (66.7)
|
80.000 (3.552-1801.650)
|
0.006
|
ADC, in situ (n=6)
|
4 (66.7)
|
25.333 (3.214-199.68)
|
0.002
|
4 (66.7)
|
39.000 (4.263-356.819)
|
0.001
|
3 (50.0)
|
40.000 (3.126-511.879)
|
0.005
|
ADC, endocervical type (n=9)
|
7 (77.8)
|
44.333 (6.230-315.499)
|
<0.001
|
8 (88.9)
|
156.000 (12.575-1935.197)
|
<0.001
|
6 (66.7)
|
80.000 (7.111-900.008)
|
<0.001
|
ADC, mucinous type (n=1)
|
0 (0.0)
|
0.000 (0.000-N/A)
|
1.000
|
0 (0.0)
|
0.000 (0.000-N/A)
|
1.000
|
0 (0.0)
|
0.000 (0.000-N/A)
|
1.000
|
Normal* (n=41)
|
3 (7.3)
|
Reference
|
-
|
2 (4.9)
|
Reference
|
-
|
1 (2.4)
|
Reference
|
-
|
ADC (n=19)
|
14 (73.7)
|
35.467 (7.475-168.274)
|
<0.001
|
14 (73.7)
|
54.600 (9.490-314.148)
|
<0.001
|
11 (57.9)
|
55.000 (6.197-488.166)
|
<0.001
|
Sensitivity (%)
|
73.7
|
73.7
|
57.9
|
Specificity (%)
|
92.7
|
95.1
|
97.6
|
PPV
|
0.824
|
0.875
|
0.917
|
NPV
|
0.884
|
0.886
|
0.833
|
* These cases only included uterine or ovarian benign diseases.
Diagnostic accuracies for ovarian or uterine tumors
The diagnostic accuracies for ovarian or endometrial tumors are listed in Table 5.
Table 5
The diagnostic accuracies of DNA methylation, hrHPV and their combination for endometrial and ovarian lesions. hrHPV, high-risk human papillomavirus. N/A, not available. OR, odds ratio. 95% CI, 95% confidence interval.
Cervical pathology
|
DNA methylation
|
hrHPV
|
DNA methylation + hrHPV
|
|
n (%)
|
OR (95% CI)
|
p
|
n (%)
|
OR (95% CI)
|
p
|
n (%)
|
OR (95% CI)
|
p
|
Normal* (n=34)
|
3 (8.8)
|
Reference
|
-
|
2 (5.9)
|
Reference
|
-
|
1 (2.9)
|
Reference
|
-
|
Ovarian benign diseases (n=7)
|
0 (0.0)
|
0.000 (0.000-N/A)
|
0.999
|
0 (0.0)
|
0.000 (0.000-N/A)
|
0.999
|
0 (0.0)
|
0.000 (0.000-N/A)
|
0.999
|
Ovarian borderline diseases (n=4)
|
0 (0.0)
|
0.000 (0.000-N/A)
|
0.999
|
0 (0.0)
|
0.000 (0.000-N/A)
|
0.999
|
0 (0.0)
|
0.000 (0.000-N/A)
|
0.999
|
Ovarian epithelial carcinomas (n=41)
|
5 (12.2)
|
1.435 (0.317-6.495)
|
0.639
|
1 (2.4)
|
0.400 (0.035-4.612)
|
0.463
|
0 (0.0)
|
0.000 (0.000-N/A)
|
0.998
|
Normal† (n=41)
|
3 (7.3)
|
Reference
|
-
|
2 (4.9)
|
Reference
|
-
|
1 (2.4)
|
Reference
|
-
|
Endometrial intraepithelial neoplasms (n=7)
|
0 (0.0)
|
0.000 (0.000-N/A)
|
0.999
|
1 (14.3)
|
3.250 (0.254-41.610)
|
0.365
|
0 (0.0)
|
0.000 (0.000-N/A)
|
0.999
|
Endometrial carcinomas (n=40)
|
22 (55.0)
|
15.481 (4.093-58.552)
|
<0.001
|
1 (2.5)
|
0.500 (0.044-5.743)
|
0.578
|
1 (2.5)
|
1.026 (0.062-16.979)
|
0.986
|
* These cases only included benign diseases except for ovarian diseases.
† These cases only included benign diseases.
Benign, borderline and malignant ovarian tumors could not be differentiated from benign diseases other than ovarian diseases by methylation assessment, hrHPV testing or their combination (all p values greater than 0.05).
In contrast to benign uterine or ovarian diseases, positive methylation was found in 55.0% (22/40) of endometrial carcinomas (p<0.001). Specifically, one case of clear cell carcinoma had positive results for methylation, while 3 cases of uterine high-grade serous cancer (HGSC) had negative results. Additionally, 7 cases of endometrioid intraepithelial neoplasia (EIN) had negative methylation results. The distributions of positive hrHPV results were similar among benign diseases, EINs and endometrial carcinomas.
Two cases of high-grade endometrial stromal sarcoma and one case of uterine malignant mixed Mullerian tumor had negative methylation results.