1Commenters’ Concerns
TZ3
postmenopausal blind spot
Referral
design bias
n=44
CIN2+ in ≥50 y
- Generalizability: colposcopy-referred cohort may inflate apparent Se/Sp vs population screening.
- Cross-sectional: no longitudinal follow-up for progression/persistence; causal inference limited.
- Precision: limited CIN2+ count (n=44) in ≥50 y subgroup widens confidence intervals.
- Overfitting: age-related epigenetic drift could confound methylation–disease links.
2Authors’ Clarifications
Pre-set
ΔCt cutoffs
NMPA
Class III validated
Pathology
biopsy/ECC endpoint
- Referral design is intentional: goal is diagnostic-support/triage in women already entering diagnostic pathways — not population screening; limitation acknowledged in Discussion.
- No model overfitting: pre-specified commercial assay with fixed ΔCt cutoffs (PAX1 ≤6.6 · JAM3 ≤10.0) validated through NMPA registration — no data-driven threshold in-cohort.
- Pathology-anchored: endpoints are colposcopy-directed biopsy/ECC (TZ3), not surrogate outcomes; conclusions limited to diagnostic/triage value.
- Methylation tracks severity: ΔCt fell with CIN grade; no HPV16/18 vs non-16/18 difference — supporting disease-severity linkage, not age artifact.
PaX1 ≤6.6
pre-set cutoff
JAM3 ≤10.0
pre-set cutoff
CV <5%
intra-assay
- No age artifact: higher methylation in older CIN2+ is framed as hypothesis-generating — consistent with prolonged HPV persistence literature.
- Agreed limitations: referral bias, lack of prior screening history capture, cross-sectional design, limited CIN2+ counts — all transparently acknowledged.
Se 93.2
CIN2+
Sp 93.6
CIN2+
Se 97.2
CIN3+
回应要点：作者认可评论者对 TZ3 绝经后盲区的判断，并强调分子分流作为补充手段的核心定位。
核心证据：CISCER CIN2+ Se 93.2%/Sp 93.6%、CIN3+ Se 97.2%——在阴道镜转诊 ≥50 岁人群中优于细胞学与指南联合筛查。
Authors concur: prospective population-based & longitudinal studies are the necessary next step to address age-related epigenetic drift and overtreatment.
3Performance Reaffirmed (≥50 y)
93.2%
CIN2+ Se
93.6%
CIN2+ Sp
97.2%
CIN3+ Se
0.934
CIN2+ AUC
- vs cytology: methylation Se 93.2% > LBC 75% and Sp 93.6% ≫ LBC 52.3% — outperforms cytology on both axes.
- vs guideline combo: higher specificity (93.6% vs 45.3%) with comparable sensitivity — fewer unnecessary colposcopies.
- Real catches: 2 hrHPV(−)/cytology(−) adenocarcinomas and high-grade lesions in non-16/18 + NILM women were methylation-positive.
4Future Directions
Population validation
Prospective & population-based cohorts with lower prevalence & different pathology spectrum.
Longitudinal risk
Follow-up to test progression/persistence & rule out age-related epigenetic drift.
Multicenter scale
Larger multicenter cohorts with HPV genotype, screening history & TZ stratification.
Health economics
Cost-effectiveness, inter-lab reproducibility & workflow fit for TZ3/ECC-limited settings.
5Clinical Positioning
- Triage, not screening: immediate utility in borderline cases — hrHPV+/cytology− or inaccessible transformation zones.
- Complementary: fills HPV/cytology gaps in older women — HPV(−) adenocarcinoma & non-16/18 high-grade lesions.
- Regulatory basis: CISCER® NMPA Class III (No. 20233400253); cutoffs pre-validated through registration.
- Transparent limits: referral bias, no systematic prior screening history, cross-sectional design, limited CIN2+ counts.
FP cut
>90% post-HPV triage
95.1%
FP reduction
90.9%
FN reduction
- Borderline triage: hrHPV+/cytology− and inaccessible-TZ women are the immediate target — where HPV/cytology are weakest.
- Reproducibility: inter-laboratory QC and standardized workflows proposed for cross-system uptake.
- Budget impact: cost-effectiveness vs local screening algorithms to be modeled in implementation research.
- Call to action: age-specific screening algorithms should incorporate molecular triage for postmenopausal women.
28/30
FP cleared 16/18
102/107
FP cleared non-16/18
2 AD
HPV(−) caught
绝经后分流价值核心：以固定阈值商业化试剂在转诊队列中提供高特异度补充诊断，减少不必要阴道镜。
Reply published in Int J Cancer 2025 — authors thank F.S. Tanveer and colleagues for constructive comments.