Immunocytochemical staining for p16, Ki-67, and MCM2 in the detection of cervical lesions and cancer: a prospective observational study

Introduction

Cervical cancer remains a major health concern worldwide, particularly in developing countries, with approximately 660,000 new cases and 348,000 related deaths occurring annually (1).

Persistent infection with high-risk human papillomavirus (HR-HPV), especially HPV types 16 and 18, is the primary cause of cervical cancer, with the other HR-HPV types including HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 (2-6).

The implementation of cervical cancer screening programs has significantly improved early detection rates, reduced mortality, and enhanced patient outcomes (7,8). However, conventional screening methods, such as the Papanicolaou (Pap) smear and HR-HPV testing, demonstrate limited sensitivity and specificity (9,10). False positives can lead to unnecessary procedures, while false negatives may involve missed diagnoses, highlighting the need for more accurate screening strategies.

Recent advances have introduced new testing options for cervical cancer screening including dual staining for p16 paired with Ki-67 (p16/Ki-67) and genotyping to specifically detect HPV types 16 and 18 in conjunction with the other HR-HPV genotypes (11,12). Additionally, novel biomarkers such as minichromosome maintenance protein 2 (MCM2) and DNA topoisomerase II α (TOP2A) have demonstrated the ability to distinguish high-risk lesions from benign abnormalities (13-15). Despite their promise, the clinical application of these biomarkers remains in the data accumulation stage.

This prospective study thus aimed to evaluate the diagnostic performance of p16, Ki-67, and MCM2 in cervical cancer screening. Their efficacy as standalone biomarkers, as elements in combination with liquid-based cytology (LBC) or HR-HPV testing, and as components of triage strategies was assessed. Our goal is to enhance the precision of early detection and contribute to the development of more effective screening approaches. We present this article in accordance with the STARD reporting checklist (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-802/rc).

Methods

Study population

Population

This prospective study included patients who underwent LBC and HR-HPV nucleic acid testing from January 1, 2023 to June 30, 2024, at the PLA General Hospital and who were referred for colposcopy and biopsy. Ethical approval was obtained from the Ethics Committee of Chinese PLA General Hospital (No. S2021-690-01), and all participants provided written informed consent. This study was conducted in accordance with the principles of the Declaration of Helsinki and its subsequent amendments, with participant confidentiality and privacy being ensured.

Inclusion criteria

The participants for this study included women aged 21–70 years with a history of sexual activity who were capable of independent decision-making and voluntarily participation. Participants were also required to have their menstrual period ended at least 3 days prior to cervical cytology sampling or tissue biopsy; to have an intact cervix with no relevant surgical history; to have undergone LBC, HR-HPV DNA/messenger RNA (mRNA) testing, and colposcopic biopsy within the previous 3 months, with the results being available; and to have a cervical exfoliated cell sample collected before colposcopy.

Exclusion criteria

Patients were excluded if they were menstruating, pregnant, or breastfeeding; had a known sexually transmitted or obvious infectious disease; or had inadequate sample collection due to issues with collection, storage, or transport.

Sample size calculation

According to the formula n=Z²p(1−p)/d², a confidence level of 95%, an expected sensitivity/specificity of 85%, and a margin of error of 5%, the required sample size was determined to be 195. To account for potential dropouts, 344 patients were enrolled, with 309 patients evaluated for p16/Ki-67 cervical type-number-strength (TNS) interpretation.

Sample collection and storage

Cervical exfoliated cell samples were collected using the Cervex-Brush device (Rovers Medical Devise, Oss, the Netherlands) and placed in SurePath vials (BD, Franklin Lakes, NJ, USA). Collection was performed before the cervical acid cytology was processed, and the samples were stored at 20–25 ℃ for less than 3 days or at 2–8 ℃ for less than 15 days. Transportation was completed with a cold pack (2–8 ℃) and did not exceed 5 days. This collection method was used for all liquid-based screening tests, which included HR-HPV, LBC, and p16 series immunocytochemical (ICC) testing, as detailed below. The residual samples were stored under the conditions recommended by the manufacturers for a period of 1–3 months, allowing for additional testing without the need for further patient visits. All screening tests were processed in accordance with standardized procedures.

HR-HPV testing

HR-HPV DNA or mRNA was detected using a commercially available kit [Polymerase Chain Reaction (PCR) Kit, Liferiver, Shanghai, China]. All assays were performed according to the manufacturer’s instructions. We detected 14 HPV types, including 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68, with genotyping performed for types 16 and 18. In this study, HR-HPV negative was categorized as no risk, types 16 and 18 were categorized as high risk, and the other 12 HR-HPV types were categorized as low risk.

Gold standard colposcopic biopsy and histology

If HPV and cytology tests produced abnormal results, colposcopy was used to examine the cervix in detail. Acetic acid was applied to highlight potential lesions, as abnormal areas turn white. Suspicious lesions identified during colposcopy were further evaluated through cervical biopsy, following the hospital’s standardized protocols. Negative for intraepithelial lesions or malignancies (NILMs) and cervical intraepithelial neoplasia (CIN) 1–2 were histologically defined as negative for cervical carcinoma, while the diagnosis of cervical carcinoma was confirmed independently by two pathologists according to the classification system of the International Federation of Gynecology and Obstetrics. In case of disagreement, a third pathologist was consulted for confirmation. In this study, according to biopsy, NILM and CIN 1–2 were categorized as negative, and CIN 3 and cervical carcinoma were categorized as positive.

LBC and p16, Ki-67, and MCM2 ICC staining

The cervical cytology samples were collected as described above and processed with the ThinPrep 2000 system (Hologic, Marlborough, MA, USA) in external laboratories in accordance with the manufacture’ protocol. The results were evaluated by a gynecological cytopathologist according to the Bethesda system (TBS) 2014 (16).

A detection kit (WECAN, Chongqing, China) was used and processed in an automated system for dual ICC staining with p16 and Ki-67 (p16/Ki-67) or p16/MCM2 proteins according to the manufacturer’s instructions. The staining was performed with residual material from the original SurePath vials collected previously. Each run included a control specimen. The slides were evaluated by a specially trained gynecological pathologist, who also assessed the original LBC slides. The results were then reviewed by another pathologist for confirmation.

The results were categorized as positive, negative, or unsatisfactory based on specific criteria for p16 (Table S1), p16/Ki-67, p16/MCM2, and p16/Ki-67/MCM2 according to the TNS + TBS interpretation standard (Tables S2-S4). The study process is summarized in Figure 1.

Figure 1 The flowchart of the study process. ICC, immunocytochemical; HPV, human papillomavirus; HR, high risk; LBC, liquid-based cytology; MCM2, minichromosome maintenance protein 2.

Statistical analysis

Data were analyzed with SPSS version 25.0 (IBM Corp., Armonk, NY, USA) and R version 4.3.0 (The R Foundation for Statistical Computing, Vienna, Austria) for Windows. Descriptive statistics of basic characteristics were analyzed via SPSS 25.0, with categorical data presented as percentages (%). Group comparisons were made via the χ² test. The sensitivity, specificity, positive predictive value, negative predictive value, and overall agreement were calculated with the “epitools” version 0.5.10 package in R. The kappa coefficient was calculated with the “psych” version 2.4.12 package in R. The overall performance of sensitivity and specificity was evaluated via the Youden index. The statistical significance was set at a P value <0.05.

Results

Basic characteristics

We recruited 344 participants from January 1, 2023 to June 30, 2024, at the PLA General Hospital. Participants were aged 21 to 70 years, with the majority (90.12%) over 30 years old. The incidence of cervical cancer and precancerous lesions increased with age. For the LBC results, those with positive histopathology results were more likely to be judged as high risk, while those with negative results were more likely to be judged as no risk and low risk. As for HPV status, the histopathologic examination produced a higher proportion of HPV16/18-positive results of 55.96%. The p16 test yielded a positivity rate of 100%. The positive rate of patients classified as high risk by the p16/Ki-67 test was 64.22%, and the positive rate of patients classified as high risk by the combined test of p16, Ki-67, and MCM2 (p16/Ki-67/MCM2) was 66.06%. p16/MCM2 high risk accounted for the majority of the positive histopathological results (85.32%). Except for age and p16, which showed no statistical differences in positive and negative results from histopathologic examination, all the other factors demonstrated significant differences (Table 1).

Diagnostic efficacy of the different screening methods

Using colposcopic biopsy results as the gold standard, we compared the diagnostic efficacy of LBC, HR-HPV, and four p16 series ICC tests (Figure 1, Table S1). The results showed that p16 ICC with a positive result as the cutoff had the highest sensitivity at 99% and the lowest specificity at 0.9%. As for the other methods, with high risk as the cutoff, we obtained a relatively higher specificity and lower sensitivity. With high risk as the cutoff, HR-HPV, the most commonly recommended screening method, had a higher specificity at 63.4% and a lower sensitivity at 56.0%. Although there were fewer unnecessary referrals for colposcopy and cervical biopsy, 3 cases of cervical cancer and 45 cases of high-grade squamous intraepithelial lesion (HSIL) were missed. In contrast, with high risk as the cutoff, we obtained relatively higher sensitivity and lower specificity. With high risk as the cutoff, HR-HPV had a higher sensitivity at 98.2%, with only case of HSIL CIN 2–3 and 1 case of cervical cancer being missed. The Youden index for LBC, HR-HPV, and the four p16 series ICC tests ranged from 0 to 0.551, and p16/MCM2, with high risk as the cutoff, yielded the highest Youden index of 0.551 (Table S5). The kappa coefficients ranged from 0 to 0.585, and the overall agreement ranged from 0.32 to 0.83. LBC, with high risk as the cutoff, had the highest kappa coefficient and overall agreement at 0.32 and 0.84, respectively (Table S5). Given the low efficacy of single-method screening, especially the recommended HR-HPV test, it may be necessary to combine two methods for screening or further triage after primary screening to increase screening efficacy.

Efficacy of the combined screening indicators in detecting early cervical cancer

We compared the diagnostic efficacy of the combinations of two methods, including of LBC, HR-HPV, and the four p16 series ICC tests (Figure 2A,2B, Table S6). The screening process is illustrated in Figure 3A. The results showed that the sensitivity and specificity of the combined screening with LBC and HR-HPV were 91.7% and 26.4%, respectively (Figure 3B,3C). Among all combinations of LBC and the p16 series ICC tests, the combination of LBC and p16/MCM2 demonstrated the highest sensitivity at 96.3% and a moderate specificity at 42.6%. We found that the biopsy results of the four participants with missed diagnoses were all HSIL CIN 2–3. Among them, only one (1/4) case was classified as low risk by both screening methods, while the other three (3/4) were identified as low risk by one method but negative by the other. In contrast, the combination of LBC and p16/Ki-67 had the lowest sensitivity at 84.31%, with the highest false-negative rate of 15.69%. Moreover, among all combinations of the HR-HPV and p16 series ICC tests, the combination of HR-HPV and p16/MCM2 had the highest sensitivity at 98.2% and a relatively lower false-negative rate of 18.7%. Notably, both participants with missed diagnoses were confirmed by biopsy as HSIL CIN 2–3 and tested positive for HR-HPV, with no cases of cervical cancer being missed.

Figure 2 Bar plots comparing the (A) sensitivity and (B) specificity of different screening methods for cervical cancer detection. Sensitivity and specificity are represented as percentages, with false-negative rates and false-positive rates stacked on top of the corresponding values. The screening methods include LBC_HR, LBC_LR, HR-HPV_HR, HR-HPV_LR, p16_positive, p16/Ki-67_HR, p16/Ki-67_LR, p16/MCM2_HR, p16/MCM2_LR, p16/Ki-67/MCM2_HR, and p16/Ki-67/MCM2_LR. HPV, human papillomavirus; HR, high risk; LBC, liquid-based cytology; LR, low risk; MCM2, minichromosome maintenance protein 2.

Figure 3 Comparison of the combined screening methods’ performance in cervical cancer detection and management. (A) A flowchart outlining the screening and management process for cervical cancer. (B) Bar plot showing the sensitivity (green bars) and false-negative rate (blue bars) of different screening methods, including LBC + HR-HPV, LBC + p16/Ki-67, LBC + p16/MCM2, LBC + p16/Ki-67/MCM2, HR-HPV + p16/Ki-67, HR-HPV + p16/MCM2, and HR-HPV + p16/Ki-67/MCM2. (C) Bar plot displaying the specificity (green bars) and false-positive rate (blue bars) of the same screening methods. HPV, human papillomavirus; HR, high risk; LBC, liquid-based cytology; MCM2, minichromosome maintenance protein 2.

The Youden index for LBC, HR-HPV, and the four p16 series ICC tests ranged from 0.17 to 0.51, with the combination of LBC and p16/Ki-67 yielding the highest Youden index of 0.51 (Table S6). The kappa coefficients ranged from 0.12 to 0.46, and the overall agreement ranged from 0.44 to 0.73. The combination of LBC and p16/Ki-67 produced the highest kappa coefficient and overall agreement, at 0.46 and 0.73, respectively.

Diagnostic efficacy of the ICC series for triage

We compared the diagnostic efficacy of the p16 series ICC test for triage after HR-HPV or LBC as primary screening in cervical cancer screening (Figure 4A-4C and Table S7). The results showed that when LBC was used as the primary screening method, and low-risk participants were further triaged via the p16 series ICC tests, p16/Ki-67/MCM2 demonstrated the highest sensitivity at 96.7%, with a specificity of 18.3%. In contrast, p16/Ki-67 had the lowest sensitivity, at 75.0%. When HR-HPV was used as the primary screening method, participants who tested positive for 12 high-risk HPV types, excluding HPV16 and HPV18, were further triaged using the p16 series ICC tests. p16/MCM2 demonstrated the highest sensitivity at 95.7%, although its specificity was only 20.2%. In contrast, p16/Ki-67 yielded a sensitivity of 80.5% and achieved the highest Youden index (0.327).

Figure 4 Comparison of screening and triage methods for cervical cancer detection and risk management. (A) Flowchart illustrating the triage and management strategies for cervical cancer based on screening results. (B) Bar plot showing the sensitivity (green bars) and false-negative rate (blue bars) of different screening and triage methods, including LBC-LR, p16/Ki-67-LR, p16/MCM2-LR, p16/Ki-67/MCM2-LR, HR-HPV-LR, p16/Ki-67-LR, p16/MCM2-LR and p16/Ki-67/MCM2-LR. (C) Bar plot displaying the specificity (green bars) and false-positive rate (blue bars) of the same screening and triage methods. HPV, human papillomavirus; HR, high risk; LBC, liquid-based cytology; LR, low risk; MCM2, minichromosome maintenance protein 2.

We also compared the overall diagnostic efficacy of HR-HPV or LBC screening with one of the p16 ICC tests as a triage method in cervical cancer screening (Table S8). The results indicated that HR-HPV primary screening with p16/MCM2 as triage had the highest sensitivity at 96.3% and the lowest false-negative rate at 3.7%. Notably, among the four cases of missed diagnosis, three were biopsy-confirmed to be HSIL CIN 2–3 and HR-HPV positive, while one was cervical cancer and HR-HPV negative. LBC screening with p16/MCM2 as triage had a similar sensitivity (80.7%) to that of HR-HPV triage (81.7%). In contrast, LBC screening with p16/Ki-67 as triage had the highest Youden index (0.48), with the sensitivity and specificity being 75.5% and 72.9%, respectively.

Discussion

Principal findings

In this study, colposcopic biopsy results were used as the gold standard for cervical cancer diagnosis. First, we assessed the performance of the screening methods used alone. The use of high risk as the cutoff yielded high sensitivity—for example, HR-HPV screening achieved a sensitivity of 98.2%—but a specificity of only 8.5%. Conversely, the use of high risk as the cutoff provided a better balance between sensitivity and specificity, with the highest Youden index observed for p16/MCM2. However, a lower sensitivity increases the risk of missed diagnoses, making it unsuitable as a standalone method. Therefore, combined screening or sequential triage is necessary to enhance screening efficacy.

HR-HPV combined with LBC or HR-HPV combined with p16/Ki-67 was effective, aligning with previous research (17). Combining HR-HPV with p16/MCM2 demonstrated higher sensitivity (98.2%) but lower specificity (18.7%), with two cases of HR-HPV-positive HSIL CIN 2–3 being missed but no cases of cervical cancer being missed. LBC combined with p16/MCM2 achieved a sensitivity of 96.3% and a specificity of 42.6%, effectively identifying high-risk individuals while reducing unnecessary referrals for colposcopy and cervical biopsy. Among the missed diagnoses, all the four cases were HSIL CIN 2–3, only one case (1/4) was classified as no risk by both screening methods, while the other three (3/4) were identified as low risk by both methods.

HR-HPV followed by p16/MCM2 triage and the combination of HR-HPV and p16-MCM2 were not significantly different in terms of specificity (19.6% vs. 18.7%) or sensitivity (96.3% vs. 98.2%) but did miss one case of cervical cancer.

Interpretation and implications

Persistent infection with high-risk HPV types, especially 16 and 18, is the primary cause of cervical cancer (3,4). A study showed that risk-based colposcopy—informed by cytology, HPV testing, and colposcopic biopsy findings—enhances the detection of cervical precancer (18). Given that most HPV infections are transient, it is essential to triage HPV-positive women to prevent unnecessary invasive procedures, such as colposcopy. HR-HPV detection and LBC are key screening methods for cervical cancer. Combining HR-HPV testing with LBC increases sensitivity and screening accuracy as compared to single methods, and these strategies allow for early detection of CIN, enabling timely intervention. Implementing comprehensive screening programs with advanced diagnostic tests and risk-based assessments is essential for preventing cervical cancer and improving prognosis (19). However, the accuracy HR-HPV testing or LBC or the combination of the two can be improved. p16/Ki-67 and MCM2 ICC can better identify high-risk cases requiring further diagnosis and reduces unnecessary colposcopies (13,20).

We found that HR-HPV testing with positive (high risk) as the cutoff demonstrated the highest sensitivity of 98.2%, indicating that it can be valuable for identifying high-risk cases; however, its low specificity (8.5%) suggests a risk of overdiagnosis and the necessity of additional combination or triage methods (11). On the other hand, we found that LBC exhibited higher specificity and lower sensitivity, indicating that relying solely on LBC could result in a substantial number of missed high-grade lesions (HSIL CIN 2–3) and even missed cervical cancer cases, aligning with the study of Woo et al. (21). These findings support a tailored, multistep screening strategy for optimizing cervical cancer detection and minimizing unnecessary interventions. Therefore, we examined the diagnostic efficacy of combining two methods or using HR-HPV or LBC for primary screening followed by another method for triage.

The study found that the combination of HR-HPV and p16/Ki-67 had a relatively high sensitivity (91.2%) and specificity (36.7%); meanwhile, using HR-HPV as the primary screening method and following it with p16/Ki-67 triage provided a sensitivity of 90.2% and a specificity of 36.7%, representing an increased specificity and similar sensitivity compared to LBC for the triage of HPV-positive women (22-24).

As for p16/MCM2, Wang et al., reported that dual staining of p16/MCM2 had higher sensitivity compared to cytological testing and better specificity compared to HPV testing. Moreover, it could identify high-grade cervical lesions and guide the classification of CIN (25). Del Moral-Hernández et al. found that dual staining of TOP2A/MCM2 was the optimal biomarker for distinguishing HSIL from low-grade squamous intraepithelial lesion (LSIL) and accounted the most cases of HSIL and cervical cancer (93.8%) as compared to p16 or Ki-67 (13). In this study, we found that p16/MCM2 alone, with high risk as cutoff, had the highest Youden index, indicating that it may be a better screening method than HR-HPV, LBC, or p16/Ki-67. Liao et al. reported that p16/MCM2 dual staining can enhance the sensitivity of cytology in a single round of screening and can predict the emergence of high-grade cervical lesions in subsequent years (26). However, p16/MCM2 combined with HR-HPV or serving as triage for HPV-positive cases had higher sensitivity than did p16/Ki-67 but only half the specificity, suggesting that it can be highly effective in detecting HSIL/CIN 2–3 lesions but susceptible to overdiagnosis. Meanwhile, HR-HPV combined with MCM2 provided no significant increase in sensitivity but had a lower specificity compared with MCM2 alone, indicating that HR-HPV with MCM2 is not an effective combination.

LBC used as the primary screening method with subsequent p16 series ICC tests as triage yielded a sensitivity lower than 85%, indicating that these methods are not suitable for the screening of cervical cancer or precancerous lesions, which is consistent with a previous study reporting that LBC is not suitable as an alternative screening method (27). However, the combination of LBC and p16/MCM2 achieved a 96.3% sensitivity and a more moderate specificity of 42.6%, which were higher than those from the combination of HR-HPV and p16/Ki-67. Furthermore, among the missed diagnoses, four cases were HSIL CIN 2–3 with no cervical cancer, only one case (1/4) was classified as no risk by both screening methods, and the other three (3/4) were identified as low risk by both methods. Thus, patients identified as low risk by either method should be recommended for a shorter interval between follow-up to achieve early detection, as supported by previous studies advocating for risk-based screening approaches that adapt follow-up strategies based on screening results (11,28).

The findings of our study highlight the potential benefits of combining various screening methods to enhance the accuracy of cervical cancer detection in clinical practice. LBC alone demonstrated suboptimal sensitivity, while HR-HPV testing exhibited low specificity, highlighting the need for more effective screening strategies. Currently, clinical guidelines recommend using HPV as the primary screening method, with Ki-67 triage for the 12 high-risk HPV types, excluding HPV16 and HPV18 (21). However, our study suggests that the combination of LBC and p16/MCM2 achieves higher sensitivity and specificity and can enhance the detection of HSIL CIN 2–3 while reducing unnecessary interventions. This approach may allow for better risk stratification, ensuring that women with true high-risk lesions are accurately identified while minimizing overtreatment in low-risk cases. Therefore, this strategy could serve as a more cost-effective alternative in cervical cancer screening.

Strengths and limitations

The prospective design employed for this study ensured robust data collection, reducing bias and enhancing reliability. By including biomarkers such as p16, Ki-67, and MCM2 alongside the established methods such as HR-HPV and LBC, we could comprehensively assess their potential in cervical cancer screening. However, despite being sufficient for initial analysis, the sample size was relatively small and should be expanded in future studies for broader validation. Moreover, the single-institution design calls for the completion of multicenter trials to assess applicability in a more diverse set of populations and healthcare settings. Future research should focus on long-term outcomes, such as CIN progression to cervical cancer and analyses of the cost-effectiveness of combining screening methods across various healthcare systems. Finally, examining alternative biomarkers and refining triage combinations could further enhance screening accuracy and efficacy.

Conclusions

Our findings suggest that HR-HPV-based primary screening with p16/Ki-67 triage may offer a viable strategy for cervical cancer detection. Combining LBC with p16/MCM2 dual staining demonstrated improved sensitivity and specificity compared to conventional methods, potentially serving as a pragmatic alternative. Notably, p16/MCM2 as a standalone assay achieved the highest Youden index when using a high-risk threshold, highlighting its promise for clinical use. Further validation through multicenter studies is essential to confirm generalizability across diverse populations and healthcare settings.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the STARD reporting checklist. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-802/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-802/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-802/prf

Funding: This work was supported by the Youth Independent Innovation Scientific Fund - Growth Project of Chinese PLA General Hospital (No. 22QNCZ017).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-802/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Ethical approval was obtained from Institutional Ethics Committee at the Chinese PLA General Hospital (No. S2021-690-01). All participants provided written informed consent. This study was conducted in accordance with the principles of the Declaration of Helsinki and its subsequent amendments, with participant confidentiality and privacy being ensured.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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(English Language Editor: J. Gray)