Fluorescence colposcope with TMTP1‐PEG4‐ICG is comparable to the conventional colposcope in identifying cervical precancerous lesions: A randomized controlled trial

To compare the diagnostic efficiency of a fluorescence colposcope with TMTP1‐PEG4‐ICG dye versus a conventional colposcope with acetic acid and Lugol's iodine in identifying cervical precancerous lesions.


| INTRODUC TI ON
Cervical cancer is the third most common female cancer worldwide.
With the improvement of screening methods and the administration of human papillomavirus (HPV) vaccines, the cervical cancer elimination plan in developed countries is accelerating. 1 More than 85% of cervical cancer deaths currently occur in low-and middle-income countries. Because of large populations, significant regional socioeconomic disparities, and insufficient medical resources, cervical cancer continues to be a serious public health problem in the developing world, including China. 2 Improving screening methods for cervical cancer is crucial. Guidelines recommend that women aged 21-29 years should perform cervical cytology test every 3 years and women aged 30-65 years should co-test with cytology and HPV every 5 years. 3 When these cervical cancer screening results are abnormal, colposcopy and colposcopy-directed biopsies are then recommended.
Colposcopy plays a pivotal role in reducing the incidence and mortality of cervical cancer. Colposcopy can provide real-time observation and evaluation of the cervix to detect cervical intraepithelial neoplasia (CIN)/squamous intraepithelial lesion (SIL) and invasive cancer. Acetic acid 3%-5% and Lugol's iodine solution are usually applied to identify potential lesions. The colposcopists give colposcopic assessment impressions according to visual changes, such as response to acetic acid (acetowhitening), characteristics of lesion borders, sizes and contours, vascular patterns, and degree of iodine uptake. 4 Cervicitis is common in colposcopy, and infected cervical cells may also become acetowhite after application of acetic acid, resulting in false-positive results. 5 Professional training and visual acuity are necessary for the screening providers. Because of the subjective nature and inherent inaccuracy of the colposcopic impression, its diagnostic efficiency varies greatly and is limited. Acetic acid and Lugol's iodine co-testing showed a sensitivity in the range 64%-100% and a specificity in the range 62%-97% in detecting CIN 2+ dysplasia. 6,7 Therefore, the development of indicators with higher diagnostic efficiency has bright application prospects.
Previously, we developed a dual modal fluorescence colposcope for simultaneous visible reflectance imaging and fluorescence imaging. The fluorescence colposcope combined with tumor-targeting near-infrared (NIR) fluorescent dye TMTP1-PEG4-ICG could help to evaluate cervical lesions in real time. 8 TMTP1-PEG4-ICG possesses advantages of increased tissue penetration and high sensitivity, and can specifically bind to cervical cancer or intraepithelial neoplasia cells, making it an ideal molecular diagnostic agent. 9 The preliminary clinical study including 11 involvers showed that the fluorescent colposcope combined with TMTP1-PEG4-ICG could display positive fluorescent signal at the lesions for squamous cell carcinoma, adenocarcinoma and CIN8.
In order to evaluate the diagnostic efficiency of this fluorescence colposcope combined with the tumor-targeting dye TMTP1-PEG4-ICG, we designed this randomized controlled trial (RCT). Patients with abnormal cervical cancer screening results were randomized to fluorescence colposcope and conventional colposcope groups, and then the diagnostic efficiencies of the two groups were compared.

| MATERIAL S AND ME THODS
The dual modal fluorescence colposcope was developed by integrating a NIR fluorescent imaging system into a conventional clinical colposcope (3ML LED; Leisegang) in our previous research. 8 The wavelength of the excitation light source was consistent with the absorption peak of TMTP1-PEG4-ICG, which was 785 nm. The At the time of the sample size calculation, there was no trials to assess the diagnostic efficiency of this fluorescence colposcope previously. However, based on the reported sensitivity and specificity of conventional colposcope in detecting cervical lesions, we calculated the sample size of the conventional colposcopy group and set the sample size of the fluorescence colposcope group to be consistent with it. We estimated a sensitivity of 88% and a specificity of 85% in the conventional colposcopy group, and set both the sensitivity and specificity error ranges to be ±0.1, and type 1 error α as 0.05.
The number of subjects required for each group was 90. Assuming a dropout rate of 10%, the total number of subjects for recruitment was set as 200.
Eligible patients were randomly divided into fluorescence colposcope and conventional colposcope groups by the random number table method. It was simple randomization: if the random number obtained was odd, the patient would be put into the fluorescence colposcope group, and if it was even, the patient would be put into the conventional colposcope group. Neither the registrar nor the colposcopy operator participated in the random grouping work. For patients of the fluorescence colposcope group, 0.1% TMTP1-PEG4-ICG fluorescent dye was applied to the cervix 30 minutes before colposcopy as previously described. 8 Two to four cervical sites with obvious fluorescent signal or suspected lesions were sampled for biopsy. For patients in the conventional colposcope group, 5% acetic acid and 5% Lugol's iodine solution were successively applied to the cervix. Two to four cervical sites with abnormal visual changes or suspected lesions were taken for biopsy. If no suspicious lesions were observed in both groups, the 3, 6, 9, 12 areas of the cervix were sampled. To avoid missed diagnosis of cervical disease, cervical canal sampling was also performed in each patient.
For conventional colposcopy, the colposcopists recorded the results of the colposcopy based on the staining of acetic acid and iodine solution and gave the colposcopic impression (normal/benign, lowgrade, high-grade, cancer) before taking the biopsy. The two operators gave colposcopic impression intraoperatively through discussion.
Colposcopy-directed biopsy was taken as the gold standard to assess  Table 1, There were no significant differences in age, Thinprep Cytologic Test (TCT), or HPV abnormalities between the two groups.
Typical images captured with the colposcopes are shown in  Table 2).
The diagnostic efficiency, including accuracy, sensitivity, specificity, PPV, NPV and associated 95% CI were calculated and are shown in Table 3 and Figure 4. The diagnostic efficiency of the fluorescence colposcope group on a per-patient basis was similar to that of the conventional colposcope group. When performing statistical analysis on a per-site basis, the NPV of the fluorescence colposcopy group was better than that of the conventional colposcopy group (97% vs. 94%, P = 0.044), and there was no significant difference in their accuracy, sensitivity, specificity and PPV. However, the difference in NPV between the fluorescence and conventional colposcope groups was not very significant, and any adjustment for multiple testing could make the P-value nonsignificant. We considered this a chance finding. These results indicated that fluorescence colposcopy and conventional colposcopy had a similar outcome in diagnosing cervical precancerous lesions.

| DISCUSS ION
In the present RCT, we found that fluorescence colposcopy with TMTP1-PEG4-ICG dye was comparable to conventional colposcopy with acetic acid and Lugol's iodine in identifying cervical precancerous lesions.
In this study, the enrollment population was not consistent with the guideline-recommended population for colposcopy. Because the TA B L E 1 Demographic characteristics.  sampling also increases the sample size, which was helpful for statistical analysis. In addition to these, we quantitatively calculated the SBRs at the lesions when evaluating the results of fluorescence colposcopy, so that the results would be more objective and convenient.
In addition to the fluorescence signal at the lesions, we could sometimes see the arc-shaped non-specific fluorescence signals at the  In this study, the sensitivity and specificity of the conventional colposcopy were 61% and 67%, respectively, which were at relatively low levels compared with previous research. 6,7 The diagnostic efficiency may also be related to the population we included. Previous studies had shown that HPV 16+ was related to clearer visual acetowhite changes in the epithelium, therefore reaching a better diagnostic efficiency for colposcopy. 19 Patients infected with any of the 16 high-risk types of HPV could be included in our study. The colposcopic impression also varied between different observers. 20 The application of dynamic spectral imaging colposcopy and related intelligent algorithms would significantly improve the diagnostic efficiency of colposcopy in the future. 21,22 This study also had some limitations and implementation chal-

F I G U R E 4
Comparison of diagnostic efficiency between the fluorescence and conventional colposcopes. NPV, negative predictive value; PPV, positive predictive value.

| CON CLUS ION
The fluorescence colposcope with TMTP1-PEG4-ICG dye was comparable to the conventional colposcope with acetic acid and Lugol's iodine in identifying cervical precancerous lesions. This novel type of fluorescence colposcopy could be a potential candidate for secondary screening of cervical cancer. However, the more convenient operation process of the fluorescence colposcope, the quality of fluorescence imaging and related intelligent algorithms need to be further improved.