Submitted:
14 April 2026
Posted:
15 April 2026
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Abstract
Background/Objectives: Tumor suppressor genes are often inactivated by genetic and epigenetic mechanisms. However, whether genetic alterations of these genes, including CDKN2A/P16, are coupled with epigenetic changes in cancer development and progression is unknown. Methods: Freshly frozen gastric carcinoma (GC) samples, paired noncancer surgical margin (SM) samples, white blood cell (WBC) samples, and clinicopathological information were collected from 200 patients. The copy number of the CDKN2A/P16 gene in these samples was determined by a P16-Light assay and normalized to that in WBCs. The DNA methylation level of the P16 promoter in GC and SM samples was determined by a 115-bp P16-specific MethyLight assay. Results: Both the P16 copy number and DNA methylation level were significantly lower in GC samples than in SM samples (median, 1.94 vs 2.14, p<0.001 for P16 CN; 0.0004 vs. 0.0013, p=0.002 for P16 methylation) and were associated with GC metastasis. The normalized P16 copy number was significantly lower in GCs without P16 methylation than in those with P16 methylation (p=0.007). Similarly, more P16 SCNdel was detected in GCs without P16 methylation than in those with P16 methylation (38.6% vs. 24.1%, p=0.027). Conclusions: Somatic P16 copy number variations are closely coupled with P16 promoter DNA methylation in the development of GC. SCNdel and promoter DNA methylation complementarily inactivate P16 in GC development and promote GC metastasis.
Keywords:
CDKN2A
; P16
; somatic copy number variation (SCNV)
; DNA methylation
; gastric cancer
; metastasis
1. Introduction
CDKN2A encodes both the P16 and P14 proteins. DNA methylation of CpG islands around the transcription start site (TSS-CGI) of the P16 gene is prevalent in precancerous lesions across organs and drives cancer development and metastasis [1,2,3,4,5,6,7,8]. Our recent findings indicate that both P16 somatic copy number deletion and amplification (SCNdel and SCNamp) are prevalent in precancerous esophageal squamous cell dysplasia (ESCdys) and noncancerous surgical margin (SM) tissues from gastric carcinoma (GC) patients. P16 SCNdel is significantly associated with poor prognosis in patients with ESCdys and GC, whereas P16 SCNamp is associated with good prognosis in patients with these diseases [9,10,11]. Both genetic and epigenetic inactivation of the P16 gene are frequent early driving events in the development of human cancers [12,13].
CGI-TSS hypermethylation not only epigenetically inactivates gene transcription [7] but also causes chromatin condensation. Because P16 inactivation leads to dysfunction of the G1-S checkpoint in the cell cycle via the RB1 pathway [14,15], RB1 loss of function by P16 inactivation may consequently cause replication stress and genome instability [16,17,18]. However, whether somatic copy number variations (SCNVs) are coupled with DNA methylation of the TSS-CGI of genes, including P16, has not been studied previously.
In this study, we compared the P16 SCNV frequency in GC and SM samples with and without P16 CGI-TSS methylation from 200 patients. We revealed for the first time that P16 SCNdel is more prevalent in gastric tissues, mainly in GCs, without P16 methylation than in those with P16 methylation, whereas P16 SCNamp is more prevalent in gastric tissues, mainly in SMs, with P16 methylation than in those without P16 methylation. P16 methylation and the SCNV complementarily promote GC development and metastasis.
2. Methods and Materials
Patients
The cohort was composed of 200 GC patients, including 106 patients with lymph metastasis and 94 patients without lymph metastasis, who underwent gastrectomy at the Peking University Cancer Hospital from January 2013 to November 2016. GC, SM (5 cm away from the main cancer mass, from the greater curvature on the distal side), and WBC samples were collected from these patients. These samples were freshly collected and stored in a freezer at -80°C at the biobank of the hospital. No cancer cells were observed in these SM samples under a microscope. Clinicopathological information and overall survival (OS) data were collected. Among these 200 patients, 80 patients were included in our previously published study, in which only P16 SCNV data, but not P16 methylation data, were available [11]. The baseline information for all 200 patients is summarized in Table 1. The Institute Review Board of the Peking University Cancer Hospital and Institute approved the study. The patients provided written informed consent to participate in this study.
Preparation of Genomic DNA
Genomic DNA was extracted from the above frozen fresh GC, SM, and WBC samples via the phenol/chloroform technique and used for P16 copy number analysis. These GC and SM DNA samples were further modified with sodium bisulfite with the EZ DNA Methylation-Gold Kit (Zymo Research) following the manufacturer's instructions and used for P16 methylation analysis.
Quantification of P16 Methylation Using the MethyLight Assay
An established 115-bp MethyLight assay [19] was used to quantify the proportion of methylated P16 alleles in triple. The COL2A1 gene, which contains no TSS-CGI, was used as an internal reference. When the copy number of methylated P16 relative to COL2A1 was greater than 2.74×10-4 in one of three PCR tubes for a bisulfite-modified DNA sample, the sample was defined as P16 methylation positive (P16M). Otherwise, it was defined as P16 methylation-negative (P16U).
Quantification of P16 Copy Number Using the P16-Light Assay
Definitions of CDKN2A/P16 SCNamp and SCNdel
As we defined previously, the average CN of P16 in WBCs from each patient was used as the diploid reference. The difference in the average P16 copy number between the tested (GC or SM) sample and the paired WBC sample was calculated for each patient. When the difference in the copy number was statistically significant (p<0.05) according to Student's t test and the absolute fold change was greater than 20%, we defined the sample as P16 SCNamp- or SCNdel-positive, as we previously reported [11].
Statistical Analysis
We used the Wilcoxon test to compare the proportion of methylated P16 alleles and Student's t test to compare P16 copy number between SM and GC samples. We used the Mann‒Whitney test to compare the proportion of methylated P16 and Student's t test or chi-square test to compare P16 copy number between different GC or SM subgroups. Both log-rank univariate and Cox multivariate analyses were used to compare patient OS between groups in the K‒M analysis. All the tests were two-sided, and a p value less than 0.05 indicated statistical significance.
3. Results
3.1. Basic results of P16 SCNV and P16 methylation analyses in GC and paired SM samples from 200 patients
The results of P16 CN and TSS-CGI methylation analyses were obtained with P16-ddPCR and MethyLight assays for GC, SM, and WBC samples from all 200 patients (Figure 1). A considerable frequency of P16 SCNamp was observed in both SMs and GCs (11.5% vs. 10.5%). Moreover, P16 SCNdel mostly occurred in GCs rather than SMs (30.5% vs. 6.5%, p<0.001) (Table 1).
The average P16 CN value was significantly greater in SMs than in GCs (Figure 2A). Similarly, the percentage of P16-methylated samples and the overall prevalence of P16 methylation were significantly greater in SMs than in GCs (81.0% vs. 56.0%, p<0.001; median, 0.0013 vs. 0.0004, p=0.002; Figure 2B), although the P16 methylation level for P16M samples was slightly lower in SMs than in GCs (median, 0.0018 vs. 0.0020; Table 2).
3.2. P16 SCNamp Coupled with P16M, Whereas P16 SCNdel Coupled with P16U in Gastric Tissues
We further compared the level of P16 CN (relative to that in WBCs) in GC and SM samples with and without P16 methylation. We found that the average P16 CN was significantly greater in P16M GC samples (n=112) than in P16U GC samples (n=88) (Figure 3A). A similar but not significant difference was also observed in the SM samples (Figure 3B). Notably, more P16 SCNdel was detected in P16U GC samples (34/88=38.6%) than in P16M GC samples (27/112=24.1%, p=0.027). These results suggest that P16 SCNdel is closely coupled with P16U, whereas P16 SCNamp is coupled with P16M in gastric cancer tissues from GC patients.
3.3. P16 SCNVs and P16M in GC or SM Samples Complementally Associated with GC Metastasis
Both the average P16 CN value and the P16 SCNamp-positive rate were significantly greater in SMs from patients without metastasis than in those from patients with metastasis (2.14 vs. 2.05, p=0.002 for P16 CN; 19.1% vs. 4.7%, p=0.004 for P16 SCNamp; Table 1).
In addition, the average P16 CN value and P16 SCNamp-positive rate were significantly greater in poorly differentiated GC samples than in well- or moderately differentiated GC samples (1.96 vs. 1.76, p=0.004 for P16 CN; 22.9% vs. 45.1%, p=0.006 for P16 SCNdel; Table 1).
A significant difference in the prevalence of P16 methylation was observed between pTNM I-II and III-IV stage GCs (0.0013 vs. 0.0036, p=0.037; Table 1). A marginally significant difference also occurred in GCs with or without lymph node metastasis (0.0025 vs. 0.0013, p=0.097; Table 1). A difference in the P16 methylation level in SMs was found between poor and well/moderately differentiated patients (0.0021 vs. 0.0013, p=0.018; Table 2).
In addition, we further investigated whether the overall survival (OS) of patients was associated with P16 SCNVs and methylation. We found that the OS of patients with P16 SCNamp-positive SMs or SCNdel-negative GCs was longer than that of patients with SCNamp-negative SMs or SCNdel-positive GCs, but the difference was not statistically significant (Figure 4A). Similar differences in OS were also observed between patients with P16 methylation-high and P16 methylation-low/no GCs (Figure 4B). In the combination analysis, no synergistic effect was detected between P16 SCNdel and methylation-high GCs or SMs (Figure 4C).
Taken together, our findings demonstrate that both P16 SCNV and methylation in gastric samples are consistently associated with GC metastasis.
4. Discussions
Tumor suppressor genes are frequently inactivated both genetically and epigenetically in cancer genomes. Inactivation of one copy of tumor suppressor genes by germline point mutations is often subsequently accompanied by epigenetic inactivation of the wild-type copy of these mutant genes by DNA methylation in adult cells and causes familial cancer [21]. For example, TSS-CGI hypermethylation serves as a frequent “2nd hit” for wild-type copies of these genes in inherited tumors and consequently causes hereditary diffuse GC and lobular breast cancer [22,23]. However, whether the SCNVs of tumor suppressor genes, including P16, are derived from TSS-CGI-methylated genes or not-methylated genes in sporadic cancer genomes is not known. Here, we report for the first time, to the best of our knowledge, that more P16 SCNdel was detected in P16U GC samples, whereas more P16 SCNamp was detected in P16U gastric samples. In addition, our findings indicate that GC metastasis is significantly associated with a decrease in P16 CN and an increase in P16 TSS-CGI methylation in gastric samples from GC patients.
Although we observed a correlation between P16 SCNamp and TSS-CGI methylation in gastric tissues from 200 patients in this study, we do not know whether P16 SCNamp and TSS-CGI methylation occur at the same alleles or within the same cells. Whether P16 SCNamp and TSS-CGI methylation are two consequent or independent events is worthy of further study.
We previously reported that P16 methylation or SCNdel increases the risk of GC metastasis [8,10,11]. In the present study, we simultaneously analyzed the states of P16 methylation and SCNVs in GC and SM samples and reported that the levels of both P16 CN and methylation correlated with GC lymph metastasis, suggesting a true role for P16 inactivation in cancer metastasis. Our findings are consistent with reports using mouse models [7,10,24].
In our previous report involving 80 GC patients [11], which was also included in the present study, we reported that the P16 SCNamp in SMs not only correlated with a low risk of GC metastasis but also correlated with long OS. However, in this study involving 200 GC patients, we observed that the P16 SCNamp in SMs correlated only with a low risk of GC metastasis, but we did not observe a significant difference in OS. The small number of patients with P16 SCNamp in SMs (23/200=11.5%) may account for the fluctuation.
5. Conclusions
P16 SCNVs are coupled with the methylation status of the P16 TSS-CGI in the development of GC. SCNdel and TSS-CGI methylation complementarily inactivate P16 and are associated with the metastasis of GC.
Author Contributions
Conceptualization, D.D.; methodology, J.Z., L.D.; validation, Z.Y. and J.Z.; formal analysis, Z.Y., J.Z., L.D.; resources, J.Q. and L,G.; data curation, Z.Y. and D.D.; writing—original draft preparation, D.D. and Z.Y.; writing—review and editing, D.D.; visualization, Z.Y. and D.D.; supervision, D.D.; project administration, D.D.; funding acquisition, D.D. All the authors have read and agreed to the published version of the manuscript.
Funding
This work was supported by the National Natural Science Foundation of China (grant number 82372586).
Institutional Review Board Statement
The Institute Review Board of the Peking University Cancer Hospital and Institute approved the study.
Informed Consent Statement
The patients provided written informed consent to participate in this study.
Data Availability Statement
The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.
Conflicts of Interest
The authors declare that they have no competing interests.
Abbreviations
The following abbreviations are used in this manuscript:
| CN | Copy number |
| ddPCR | Droplet digital PCR |
| ESCdys | esophageal squamous cell dysplasia |
| GC | gastric carcinoma |
| OS | overall survival |
| P16M | P16 methylation positive |
| P16U | P16 methylation negative |
| SCNamp | somatic copy number amplification |
| SCNdel | somatic copy number deletion |
| SCNVs | somatic copy number variation |
| SM | surgical margin |
| TSS-CGI | CpG islands around transcription starting site |
| WBC | white blood cell |
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Figure 1.
Detailed results of P16 SCNV and methylation analyses for GC and SM samples from all 200 patients with and without metastasis. The exact differences in P16 CNs between WBCs and GCs or SMs and P16 methylation levels are listed for each GC or SM sample. SCNamp or SCNdel, somatic copy number amplification or deletion; P16M or P16U, P16 TSS-CGI methylation-positive or -negative.
Figure 1.
Detailed results of P16 SCNV and methylation analyses for GC and SM samples from all 200 patients with and without metastasis. The exact differences in P16 CNs between WBCs and GCs or SMs and P16 methylation levels are listed for each GC or SM sample. SCNamp or SCNdel, somatic copy number amplification or deletion; P16M or P16U, P16 TSS-CGI methylation-positive or -negative.
Figure 2.
Comparison of P16 CN and TSS-CGI methylation levels in GC and paired SM samples from 200 patients. (A) P16 CN level (relative to WBC); (B) copy number of methylated P16 (relative to COL2A1); GC and paired SM samples from the same patient are linked with a black line. The average P16 CN and P16 methylation values (median) are labeled.
Figure 2.
Comparison of P16 CN and TSS-CGI methylation levels in GC and paired SM samples from 200 patients. (A) P16 CN level (relative to WBC); (B) copy number of methylated P16 (relative to COL2A1); GC and paired SM samples from the same patient are linked with a black line. The average P16 CN and P16 methylation values (median) are labeled.
Figure 3.
Comparison of differences in P16 CN between gastric tissue samples with and without P16 methylation from 200 patients. (A) Percentage change in the P16 CN in GCs relative to that in WBCs; (B) percentage change in the P16 CN in SMs relative to that in WBCs; the SCNV types are labeled with different colors. P16M and P16U, P16 TSS-CGI methylation-positive and -negative.
Figure 3.
Comparison of differences in P16 CN between gastric tissue samples with and without P16 methylation from 200 patients. (A) Percentage change in the P16 CN in GCs relative to that in WBCs; (B) percentage change in the P16 CN in SMs relative to that in WBCs; the SCNV types are labeled with different colors. P16M and P16U, P16 TSS-CGI methylation-positive and -negative.
Figure 4.
OS curves for GC patients with different states of P16 SCNVs and TSS-CGI methylation in gastric tissues according to K‒M analyses. (A) OS curves for patients with and without P16 SCNdel in GCs or SCNamp in SMs; (B) OS curves for patients with and without P16 methylation-high in GCs or SMs; (C) OS curves for patients with and without P16 SCNdel &/or methylation-high in GCs or SMs. The hazard ratio (HR), 95% CI, and p value are labeled according to log-rank univariate analysis.
Figure 4.
OS curves for GC patients with different states of P16 SCNVs and TSS-CGI methylation in gastric tissues according to K‒M analyses. (A) OS curves for patients with and without P16 SCNdel in GCs or SCNamp in SMs; (B) OS curves for patients with and without P16 methylation-high in GCs or SMs; (C) OS curves for patients with and without P16 SCNdel &/or methylation-high in GCs or SMs. The hazard ratio (HR), 95% CI, and p value are labeled according to log-rank univariate analysis.
Table 1.
Prevalence of somatic copy number variations (SCNVs) of the P16 gene in GC and SM samples from 200 patients.
Table 1.
Prevalence of somatic copy number variations (SCNVs) of the P16 gene in GC and SM samples from 200 patients.
| GC | SM | |||||||||||||
| P16 CNa | P16 CN | Case number for P16 SCNV (%) | ||||||||||||
| n | Mean±SD | p valuec | SCNdel | Diploid | SCNamp | p valued | Mean±SD | p value | SCNdel | Diploid | SCNamp | p value | ||
| Age (yr) | ≤60 | 80 | 1.95 ± 0.41 | 0.164 | 20 (25.0) | 49 (61.3) | 11 (13.7) | 0.249 | 2.08 ± 0.23 | 0.508 | 6 (7.5) | 65 (81.3) | 9 (11.2) | 0.895 |
| >60 | 120 | 1.86 ± 0.48 | 41 (34.2) | 69 (57.5) | 10 (8.3) | 2.10 ± 0.22 | 7 (5.8) | 99 (82.5) | 14 (11.7) | |||||
| Sex | Male | 140 | 1.89 ± 0.48 | 0.924 | 44 (31.4) | 81 (57.9) | 15 (10.7) | 0.880 | 2.10 ± 0.22 | 0.833 | 8 (5.7) | 115 (82.1) | 17 (12.2) | 0.736 |
| Female | 60 | 1.90 ± 0.39 | 17 (28.3) | 37 (61.7) | 6 (10.0) | 2.09 ± 0.22 | 5 (8.3) | 49 (81.7) | 6 (10.0) | |||||
| Neoadjuvant | Yes | 64 | 1.95 ± 0.45 | 0.193 | 17 (26.6) | 37 (57.8) | 10 (15.6) | 0.240 | 2.09 ± 0.27 | 0.857 | 4 (6.3) | 49 (76.6) | 11 (17.1) | 0.224 |
| chemotherapy | No | 136 | 1.86 ± 0.46 | 44 (32.4) | 81 (59.6) | 11 (8.0) | 2.09 ± 0.19 | 9 (6.6) | 115 (84.6) | 12 (8.8) | ||||
| GC location | Noncardiac | 141 | 1.92 ± 0.43 | 0.192 | 41 (29.1) | 84 (59.6) | 16 (11.3) | 0.715 | 2.09 ± 0.23 | 0.635 | 10 (7.1) | 116 (82.3) | 15 (10.6) | 0.752 |
| Cardiac | 59 | 1.83 ± 0.51 | 20 (33.9) | 34 (57.6) | 5 (8.5) | 2.10 ± 0.21 | 3 (5.1) | 48 (81.4) | 8 (13.5) | |||||
| Differentiatione | Well or mod. | 71 | 1.76 ± 0.50 | 0.004 | 32 (45.1) | 32 (45.1) | 7 (9.8) | 0.006 | 2.07 ± 0.27 | 0.456 | 7 (9.9) | 54 (76.1) | 10 (14.0) | 0.186 |
| Poor | 118 | 1.96 ± 0.43 | 27 (22.9) | 77 (65.3) | 14 (11.8) | 2.10 ± 0.18 | 6 (5.1) | 102 (86.4) | 10 (8.5) | |||||
| pTNM stage | I-II | 117 | 1.91 ± 0.45 | 0.558 | 34 (29.1) | 71 (60.7) | 12 (10.2) | 0.842 | 2.12 ± 0.23 | 0.020 | 8 (6.8) | 89 (76.1) | 20 (17.1) | 0.012 |
| III-IV | 83 | 1.87 ± 0.46 | 27 (32.5) | 47 (56.6) | 9 (10.9) | 2.05 ± 0.21 | 5 (6.0) | 75 (90.4) | 3 (3.6) | |||||
| Local invasion | T1-2 | 45 | 1.86 ± 0.46 | 0.573 | 17 (37.8) | 24 (53.3) | 4 (8.9) | 0.480 | 2.10 ± 0.26 | 0.706 | 6 (13.3) | 32 (71.1) | 7 (15.6) | 0.054 |
| T3-4 | 155 | 1.90 ± 0.46 | 44 (28.4) | 94 (60.6) | 17 (11.0) | 2.09 ± 0.21 | 7 (4.5) | 132 (85.2) | 16 (10.3) | |||||
| Lymph | N0 | 94 | 1.91 ± 0.45 | 0.589 | 26 (27.7) | 58 (61.7) | 10(10.6) | 0.708 | 2.14 ± 0.22 | 0.002 | 4 (4.3) | 72 (76.6) | 18 (19.1) | 0.004 |
| metastasis | N1-Xf | 106 | 1.88 ± 0.47 | 35 (33.0) | 60 (56.6) | 11 (10.4) | 2.05 ± 0.21 | 9 (8.5) | 92 (86.8) | 5 (4.7) | ||||
| (total) | 200 | 1.89 ± 0.46 | 61 (30.5)g | 118 (59.0) | 21 (10.5) | 2.09 ± 0.22 | 13 (6.5) | 164 (82.0) | 23 (11.5) | |||||
aP16 copy number (CN) in GC or SM was adjusted by that of WBC from the same patient; b somatic copy number variations of P16 relative to WBC from the same patient; SCNdel or SCNamp: difference in P16 CN between WBC and tested tissue is ≤80% or ≥120% and p<0.05 according to Student's t test; c Student's t test; d chi-square test; e no differentiation information for 11 cases; f including 7 cases with distant metastasis; g GC vs. SM, p<0.001
Table 2.
The prevalence of P16 methylation in gastric adenocarcinoma (GC) and surgical margin (SM) tissue samples from 200 patients.
Table 2.
The prevalence of P16 methylation in gastric adenocarcinoma (GC) and surgical margin (SM) tissue samples from 200 patients.
| Prevalence of p16 methylation | ||||||||
| GC | SM | |||||||
| n | Positive rate (%) | Methylation level (median, 25-75%) a,b | p valuec | Positive rate (%) | Methylation level (median, 25-75%) | p valuec | ||
| Age (yr) | ≤60 | 80 | 52 (65.0) | 0.20 (0.08-0.71) | 0.580 | 69 (86.3) | 0.18 (0.07-0.37) | 0.524 |
| >60 | 120 | 60 (50.0) | 0.20 (0.07-2.19) | 93 (77.5) | 0.19 (0.07-0.39) | |||
| Sex | Male | 140 | 78 (55.7) | 0.14 (0.07-1.48) | 0.437 | 111 (79.3) | 0.17 (0.07-0.33) | 0.164 |
| Female | 60 | 34 (56.7) | 0.34 (0.08-0.98) | 51 (85.0) | 0.23 (0.07-0.62) | |||
| Neoadjuvant | Yes | 64 | 33 (51.6) | 0.12 (0.06-0.52) | 0.293 | 48 (75.0) | 0.14 (0.05-0.27) | 0.011 |
| chemotherapy | No | 136 | 79 (58.1) | 0.32 (0.08-1.47) | 114 (83.8) | 0.21 (0.09-0.60) | ||
| GC location | Noncardiac | 141 | 85 (60.3) | 0.20 (0.08-0.99) | 0.724 | 119 (84.4) | 0.19 (0.07-0.42) | 0.383 |
| Cardiac | 59 | 27 (45.8) | 0.12 (0.07-3.79) | 43 (72.9) | 0.14 (0.06-0.31) | |||
| Differentiation | Well or mod. | 71 | 30 (42.3) | 0.19 (0.08-1.65) | 0.873 | 56 (78.9) | 0.13 (0.06-0.29) | 0.018 |
| Poor | 118 | 75 (63.6) | 0.20 (0.07-1.32) | 99 (83.9) | 0.21 (0.11-0.40) | |||
| pTNM stage | I-II | 117 | 64 (54.7) | 0.13 (0.06-0.51) | 0.037 | 90 (76.9) | 0.21 (0.07-0.37) | 0.950 |
| III-IV | 83 | 48 (57.8) | 0.36 (0.08-2.57) | 72 (86.7) | 0.17 (0.07-0.52) | |||
| Local invasion | T1-2 | 45 | 25 (55.6) | 0.13 (0.06-1.14) | 0.663 | 39 (86.7) | 0.21 (0.07-0.37) | 0.842 |
| T3-4 | 155 | 87 (56.1) | 0.20 (0.08-1.08) | 123 (79.4) | 0.18 (0.07-0.39) | |||
| Lymph | N0 | 94 | 51 (54.3) | 0.13 (0.07-0.52) | 0.097 | 70 (74.5) | 0.23 (0.07-0.37) | 0.627 |
| metastasis | N1-X | 106 | 61 (57.5) | 0.25 (0.07-2.56) | 92 (86.8) | 0.16 (0.07-0.52) | ||
| (total) | 200 | 112 (56.0) d | 0.20 (0.07-1.06) | 162 (81.0) | 0.18 (0.07-0.37) | |||
a for P16M samples; b ×10-2; c Mann‒Whitney test; d GC vs. SM, p=0.002.
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